Systems and methods for producing stem cells and differentiated cells

ABSTRACT

The present invention provides various improved systems and methods for obtaining, generating, culturing, and handling cells, such as stem cells (including induced pluripotent stem cells or iPSCs) and differentiated cells, as well as cells and cell panels produced using such systems and methods, and uses of such cells and cell panels.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application U.S. application Ser.No. 16/587,801 filed Sep. 30, 2019, which is a continuation ofapplication of U.S. application Ser. No. 14/318,339 filed Jun. 27, 2014,now issued as U.S. Pat. No. 10,428,309; which is a continuation-in-partapplication of U.S. application Ser. No. 13/691,258 filed Nov. 30, 2012,now issued as U.S. Pat. No. 10,273,459; which claims the benefit under35 U.S.C. § 119(e) to U.S. Application Ser. No. 61/700,792 filed Sep.13, 2012, U.S. Application Ser. No. 61/580,007 filed Dec. 23, 2011 andU.S. Application Ser. No. 61/565,818 filed Dec. 1, 2011, all nowexpired. U.S. application Ser. No. 14/318,339 filed Jun. 27, 2014, nowissued as U.S. Pat. No. 10,428,309, also claims the benefit under 35U.S.C. § 119(e) to U.S. Application Ser. No. 61/840,271 filed Jun. 27,2013, now expired. The disclosure of each of the prior applications isconsidered part of and is incorporated by referenced in the disclosureof this application.

REFERENCE TO A SEQUENCE LISTING

The material in the accompanying Sequence Listing is hereby incorporatedby reference into this application. The accompanying sequence listingtext file named NYS_002CPC2_SEQ.txt, was created on Sep. 7, 2021 and is5 KB. The file can be accessed using Microsoft Word on a computer thatuses Windows OS.

A portion of the disclosure of this patent document contains materialthat is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

BACKGROUND OF THE INVENTION

Stem cells are unspecialized cells that self-renew for long periodsthrough cell division, and can be induced to differentiate into cellswith specialized functions, i.e., differentiated cells. These qualitiesgive stem cells great promise for use in therapeutic applications toreplace damaged cells and tissue in various medical conditions.Embryonic stem (ES) cells are derived from the blastocyst of an earlystage embryo and have the potential to develop into endoderm, ectoderm,and mesoderm (the three germ layers) (i.e., they are “pluripotent”). Invitro, ES cells tend to spontaneously differentiate into various typesof tissues, and the control of their direction of differentiation can bechallenging. There are unresolved ethical concerns that are associatedwith the destruction of embryos in order to harvest human ES cells.These problems limit their availability for research and therapeuticapplications.

Adult stem (AS) cells are found among differentiated tissues. Stem cellsobtained from adult tissues typically have the potential to form a morelimited spectrum of cells (i.e., “multipotent”), and typically onlydifferentiate into the cell types of the tissues in which they arefound, though recent reports have shown some plasticity in certain typesof AS cells. They also generally have a limited proliferation potential.

Induced pluripotent stem cells (iPSC or iPSCs) are produced bylaboratory methods from differentiated adult cells. iPSCs are widelyrecognized as important tools, e.g., for conducting medical research.Heretofore, the technology for producing iPSCs has been time-consumingand labor-intensive. Differentiated adult cells, e.g., fibroblasts, arereprogrammed, cultured, and allowed to form individual colonies whichrepresent unique clones. Previously, identifying these types of cellshas been difficult because the majority of the cells are notfully-reprogrammed iPSC clones. The standard is for iPSC clones to beselected based on the morphology of the cells, with desirable coloniespossessing sharply demarcated borders containing cells with a highnuclear-to-cytoplasmic ratio. When clones are identified, they aremanually-picked by micro-thin glass tools and cultured on “feeder”layers of cells typically, murine embryonic fibroblasts (MEFs). Thisstep is performed typically at 14-21 days post-infection with areprograming vector. Then the clones are expanded for another 14-21 daysor more, prior to undergoing molecular characterization.

Others have focused on developing techniques to rapidly and moreaccurately identify and characterize fully-reprogrammed adultfibroblasts and their downstream differentiation potential (Bock et al.,2011, Cell 144: 439-452; Boulting et al., 2011, Nat Biotechnol 29:279-286). Also see, for example, co-owned U.S. Ser. No. 13/159,030,filed on Jun. 13, 2011, describing the use of Fluorescence ActivatedCell Sorting (FACS) to identify and live sort unique subpopulations ofstem cells as defined by unique expression patterns of surface proteins.

Thus, stem cells are an attractive source of cells for therapeuticapplications, medical research, pharmaceutical testing, and the like.However, there remains a longstanding need in the art for an automatedsystem for rapidly producing and isolating reproducible iPSC cell linesunder standard conditions in order to meet these and other needs. Therealso there remains in the art a need for methods of making panels ofiPSC cell lines, and differentiated cells produced therefrom, that arederived from multiple different individuals in a “population ofinterest.”

SUMMARY OF THE INVENTION

The present invention provides various improved systems and methods forobtaining, generating, culturing, and handling cells, such as stem cells(including induced pluripotent stem cells or iPSCs) and differentiatedcells, as well as cells and cell panels produced using such systems andmethods, and uses of such cells and cell panels. The present inventionbuilds upon, and provides certain improvements over, the systems andmethods described previously in international patent applicationPCT/US2012/067417 (published on Jun. 6, 2013 with publication numberWO/2013/082509) and U.S. patent application Ser. No. 13/691,258(published on Dec. 26, 2013 with publication number 2013-0345094), thecontents of each of which are hereby incorporated by reference in theirentireties for those jurisdictions that permit incorporation byreference. Building on the work described in these prior patentapplications, the Applicants have now developed certain improved methodsand systems that can significantly reduce variability in obtaining,generating, culturing, and handling a variety of cell types, includingdifferentiated cells and stem cells. Such improved systems and methodsare particularly advantageous for automated and/or high-throughputapplications—greatly facilitating the ability to work with large numbersof cells in a parallel manner.

For example, the Applicants have discovered that variability in thegeneration and culture and differentiated cells and stem cells can bereduced, allowing for more efficient parallel processing, if individualsamples are selected and grouped according to various characteristics(such as growth characteristics or donor age), such that cells havingsimilar characteristics are grown and handled together. In some parts ofthis application such methods may be referred to as “binning” methods,or “batching” methods, or “binning and batching” methods, or“data-driven batching” methods. Applicants also discovered that theefficiency of cellular reprograming could be improved, and thevariability in reprogramming efficiency reduced, when cells were grownin low serum or serum free medium for a certain amount of time prior to,and optionally also during, reprogramming. This finding was unexpectedgiven the widely-held view that growth rates should be maximized forreprogramming. Indeed, consistent with this prevailing view, cells aretypically grown in high serum media (e.g., containing 10% serum) priorto reprogramming and then subsequently switched to low serum or serumfree medium at a later stage. While the Applicants, like others, foundthat increased growth rates were beneficial for reprogramming,Applicants' findings suggested that switching cells to low serum mediumat the time of, or soon after, reprogramming may shock the cells andhave a detrimental effect on reprogramming success. Applicants foundthat such effects could be mitigated by allowing the cells to adjust tolow serum conditions for a certain amount of time prior toreprogramming. Applicants also found that differences in methods used togenerate embryoid bodies (EBs) from pluripotent stem cells hadsignificant and surprising effects on differentiation. For example,Applicants found that “hanging drop” methods for EB generation led to abias towards endoderm differentiation, while the generation of EBs inV-bottom plates led to a more uniform differentiation potential, as wellwe being better suited to automated and high-throughput systems. Theseand other improvements to systems and methods for obtaining, generating,culturing and handling differentiated cells and stem cells, aredescribed in further detail in this Summary of the Invention section, aswell as throughout other sections of this patent application, includingthe Detailed Description, Examples, Drawings & Claims.

As mentioned above, Applicants have discovered that variability in thegeneration and culture of differentiated cells and stem cells can bereduced if individual samples are selected and grouped according tovarious characteristics, such that cells having similar characteristicsare grown and handled together. Such methods can reduce variability inthe generation and culture and differentiated cells and stem cells,which is particularly advantageous in applications requiring parallelprocessing of multiple different samples, such as in automated and/orhigh-throughput methods. Similarly, the ability to select, group andhandle samples according to various particular characteristics may beuseful for the generation of cell panels for use in a variety ofapplications, including for use in drug screening. The present inventionprovides a variety of methods and systems that can be used to select andgroup samples in such ways. Such methods and systems may be referred toherein as data-driven batching methods or systems. Accordingly, in oneembodiment the present invention provides a method for the automatedgeneration and/or manipulation of stem cells, the method comprising: (a)culturing multiple different samples of cells, wherein the cellscomprise adult somatic cells or induced pluripotent stem cells, (b)determining the proliferation rate or cell doubling time of individualcell samples from among the multiple different samples of cells, (c)freezing individual cell samples from among the multiple differentsamples of cells, (d) selecting from the individual cell samples asubset of samples having similar proliferation rates or cell doublingtimes, (e) thawing the subset of samples selected in step (d), (f)plating the subset of samples in a multi-well plate, (g) culturing thesubset of samples until they reach a desired confluency, and (h) wherethe cells comprise adult somatic cells, contacting the somatic cellswith one or more reprogramming factors in order to produce iPSCs, or,where the cells comprise iPSCs, treating the iPSCs in order to producedifferentiated cells. Similarly, in one embodiment the present inventionprovides a method for the efficient generation, culture and/or handlingof multiple cell samples in parallel, the method comprising: (a)culturing multiple different cell samples, (b) determining, or obtaininginformation regarding, one or more characteristics or properties ofindividual samples from among the multiple different cell samples (or ofthe donor/subject from which such samples were obtained), (c) selectingfrom the multiple different cell samples a subset of cell samples havinga desired characteristic or property, (d) plating the subset of cellsamples in a multi-well plate, and (e) culturing the subset of cellsamples in the multi-well plate. Similarly, in other embodiment, thepresent invention provides a method for the efficient culture ofmultiple cell samples in parallel, the method comprising: (a) culturingmultiple different cell samples, (b) determining, or obtaininginformation regarding, one or more characteristics or properties ofindividual samples from among the multiple different cell samples (or ofthe donor/subject from which such samples were obtained), (c) freezingindividual cell samples from among the multiple different cell samples,(d) selecting from the multiple different cell samples a subset of cellsamples having a desired characteristic or property, (e) thawing thesubset of cell samples selected in step (d) plating the subset of cellsamples in a multi-well plate, and (f) culturing the subset of cellsamples in the multi-well plate. In some such embodiments thecharacteristic or property is the cellular proliferation rate or celldoubling time of a cell sample. In some such embodiments, thecharacteristic or property relates to the age, sex, race, ethnicity,diagnosis (e.g., for a disease or a disorder), genotype, phenotype,blood type, HLA type, treatment history, or drug response profile of thecell sample or of the donor/subject from which the cell sample wasobtained. In some such embodiments cell samples are selected on thebasis of at least two characteristics, including (i) the cellularproliferation rate or cell doubling time of the cell sample, and (ii)the age, sex, race, ethnicity, diagnosis (e.g., for a disease or adisorder), genotype, phenotype, blood type, HLA type, treatment history,or drug response profile of the cell sample or the donor/subject fromwhich the cell sample was obtained. In some such embodiments the cellsamples are adult somatic cells, such as adult somatic fibroblasts. Insome such embodiments the cell samples are pluripotent stem cells, suchas induced pluripotent stem cell (iPSCs). In some such embodiments thecell samples are differentiated cells derived from pluripotent stemcells. In some such embodiments one or more of the steps is automated.In some embodiments, each of the steps is automated. In some suchembodiments the step of selecting from the multiple different cellsamples a subset of the cell samples is automated and/or performed by acomputer. In some such embodiments, where samples are selected based onhaving similar proliferation rates or cell doubling times, the selectedsamples have proliferation rates or cell doubling times that vary byless than 30% between samples, or by less than 25% between samples, orby less than 20% between samples, or by less than 15% between samples,or by less than 10% between samples, or by less than 5% between samples,or by less than 2% between samples.

In another embodiment, the present invention provides a method for theefficient generation of induced pluripotent stem cells fromdifferentiated adult somatic cells, the method comprising: (a) culturingmultiple different samples of differentiated adult somatic cells, (b)determining the proliferation rate or cell doubling time of individualsamples from among the multiple different samples of differentiatedadult somatic cells, (c) selecting from the multiple different samplesof differentiated adult somatic cells a subset of samples having similarproliferation rates or cell doubling times, (d) plating the subset ofthe samples selected in a multi-well plate, such that each of thesamples in the multi-well plate has a similar proliferation rate or celldoubling time, (e) culturing the cell samples in the multi-well plateuntil they reach a desired confluency, and (f) contacting the cellsamples with one or more reprogramming factors in order to produceiPSCs. Similarly, in another embodiment, the present invention providesa method for the automated generation of induced pluripotent stem cellsfrom differentiated adult somatic cells, the method comprising: (a)culturing multiple different samples of differentiated adult somaticcells, (b) determining the proliferation rate or cell doubling time ofindividual samples from among the multiple different samples ofdifferentiated adult somatic cells, (c) freezing individual cell samplesfrom among the multiple different cell samples, (d) selecting from themultiple different samples of differentiated adult somatic cells asubset of the samples having similar proliferation rates or celldoubling times, (e) thawing and plating the subset of the samplesselected in step (d) into a multi-well plate, such that each of thesamples in the multi-well plate has a similar proliferation rate or celldoubling time, (f) culturing the cell samples in the multi-well plateuntil they reach a desired confluency, and (g) contacting the cellsamples with one or more reprogramming factors in order to produceiPSCs. In some such embodiments the differentiated adult somatic cellsare fibroblasts. In some such embodiments the fibroblasts are derivedfrom human donors/subjects. In some such embodiments one or more of thesteps is automated. In some such embodiments, each of the steps isautomated. In some such embodiments the step of selecting from themultiple different cell samples a subset of the cell samples havingsimilar proliferation rates or cell doubling times is performed by acomputer. In some such embodiments, where samples are selected based onhaving similar proliferation rates or cell doubling times, the selectedsamples have proliferation rates or cell doubling times that vary byless than 30% between samples, or by less than 25% between samples, orby less than 20% between samples, or by less than 15% between samples,or by less than 10% between samples, or by less than 5% between samples,or by less than 2% between samples. In some such embodiments theculturing of the somatic cells prior to contacting with reprogrammingfactors is performed in low serum medium or in serum free medium. Insome such embodiments the step of selecting a subset of the cell samplesfurther comprises selecting cell samples on the basis of the age, sex,race, ethnicity, diagnosis (e.g., for a disease or a disorder),genotype, phenotype, blood type, HLA type, treatment history, or drugresponse profile of the cell sample or the donor/subject from which thecell sample was obtained. In some such embodiments the method furthercomprises producing differentiated cells from the pluripotent stemcells, for example by contacting the pluripotent stem cells with one ormore differentiation factors or by generating embryoid bodies (EBs). Insome such embodiments, where EBs are generated from pluripotent stemcells, the EBs are generated in V-bottom plates.

In one embodiment, the present invention provides a method for theefficient generation of human induced pluripotent stem cells (iPSCs)from human donor fibroblasts, the method comprising: (a) culturingmultiple different samples of human donor fibroblasts, (b) determiningthe proliferation rate or cell doubling time of individual samples fromamong the multiple different samples of human donor fibroblasts, (c)freezing individual cell samples from among the multiple differentsamples of human donor fibroblasts, (d) selecting from the multipledifferent samples of human donor fibroblasts a subset of the sampleshaving similar proliferation rates or cell doubling times, (e) thawingthe subset of samples selected in step (d), (f) culturing the subset ofcell samples in a multi-well plate, such that each of the samples in themulti-well plate has a similar proliferation rate or cell doubling time,wherein the culturing comprises contacting the cell samples with lowserum medium for at least 3 days, and (g) subsequently contacting thecell samples with one or more reprogramming factors in order to produceiPSCs. In one such embodiment one or more of the steps is automated. Inone such embodiment each of the steps is automated. In one suchembodiment the step of selecting from the multiple different cellsamples a subset of the cell samples having similar proliferation ratesor cell doubling times is automated and/or performed by a computer. Insome such embodiments, where samples are selected based on havingsimilar proliferation rates or cell doubling times, the selected sampleshave proliferation rates or cell doubling times that vary by less than30% between samples, or by less than 25% between samples, or by lessthan 20% between samples, or by less than 15% between samples, or byless than 10% between samples, or by less than 5% between samples, or byless than 2% between samples. In one such embodiments the step ofselecting a subset of the cell samples further comprises selecting cellsamples on the basis of the age, sex, race, ethnicity, diagnosis (e.g.,for a disease or a disorder), genotype, phenotype, blood type, HLA type,treatment history, or drug response profile of the cell sample or thedonor/subject from which the cell sample was obtained.

In one embodiment, the present invention provides a method for theautomated generation of differentiated cells from pluripotent stemcells, the method comprising: (a) culturing multiple different samplesof pluripotent stem cells, (b) determining the proliferation rate orcell doubling time for individual samples from among the multipledifferent samples of pluripotent stem cells, (c) selecting from themultiple different samples of pluripotent stem cells a subset of sampleshaving similar proliferation rates or cell doubling times, (d) platingthe subset of the samples selected in step (c) into a multi-well plate,such that each of the samples in the multi-well plate has a similarproliferation rate or cell doubling time, (e) culturing the cell samplesin the multi-well plate until they reach a desired passage number and/orconfluency, and (f) producing differentiated cells from the pluripotentstem cells. Similarly, in another embodiment, the present inventionprovides a method for the automated generation of differentiated cellsfrom pluripotent stem cells, the method comprising: (a) culturingmultiple different samples of pluripotent stem cells, (b) determiningthe proliferation rate or cell doubling time for each of the multipledifferent samples of pluripotent stem cells, (c) freezing individualcell samples from among the multiple different cell samples, (d)selecting from the multiple different samples of pluripotent stem cellsa subset of the samples having similar proliferation rates or celldoubling times, (e) thawing and plating the subset of the samplesselected in step (x) into a multi-well plate, such that each of thesamples in the multi-well plate has a similar proliferation rate or celldoubling time, (f) culturing the cell samples in the multi-well plateuntil they reach a desired passage number and/or confluency, and (g)producing differentiated cells from the pluripotent stem cells. In somesuch embodiments the pluripotent stem cells are induced the pluripotentstem cells (iPSCs). In some such embodiments one or more of the steps isautomated. In some such embodiments the each of the steps is automated.In some such embodiments the step of selecting from the multipledifferent cell samples a subset of the cell samples having similarproliferation rates or cell doubling times is automated and/or performedby a computer. In some such embodiments, where samples are selectedbased on having similar proliferation rates or cell doubling times, theselected samples have proliferation rates or cell doubling times thatvary by less than 30% between samples, or by less than 25% betweensamples, or by less than 20% between samples, or by less than 15%between samples, or by less than 10% between samples, or by less than 5%between samples, or by less than 2% between samples. In some suchembodiments, the step of producing differentiated cells from the iPSCscomprises contacting the iPSCs with one or more differentiation factor.In some such embodiments the step of producing differentiated cells fromthe iPSCs comprises generating embryoid bodies (EBs). In some suchembodiments the step of producing differentiated cells from the iPSCscomprises generating embryoid bodies (EBs) in V-bottom plates. In somesuch embodiments the step of selecting a subset of the cell samplesfurther comprises selecting cell samples on the basis of the age, sex,race, ethnicity, diagnosis (e.g., for a disease or a disorder),genotype, phenotype, blood type, HLA type, treatment history, or drugresponse profile of the cell sample or the donor/subject from which thecell sample was obtained.

The present invention also provides automated “data-driven batchingsystems” that can be used to select and group (or “bin and/or batch”)cell samples based on one or more properties or characteristics, asdescribed above. Such automated data-driven batching systems systems canbe used, for example, to select and group somatic cells (such asfibroblasts) for analysis or for subsequent iPSC generation, to selectand group stem cells (such as iPSCs) for analysis or for subsequentdifferentiation, and/or to select and group cells (such as somatic cellsobtained from donors, iPSCs, or differentiated cells derived from iPSCs)for inclusion in a cell panel. The components of the data-drivenbatching systems described here can comprise, or can be modified from,or can be used in conjunction with, the other automated systems, andcomponents thereof, described herein and/or those described ininternational patent application PCT/US2012/067417 and U.S. patentapplication Ser. No. 13/691,258. Further, one of skill in the art willappreciate where and how the automated systems described herein (and inPCT/US2012/067417 and U.S. Ser. No. 13/691,258) can be modified toprovide or include such data-driven batching systems.

For example, in some embodiments, the present invention provides anautomated data-driven batching system that comprises: (a) acomponent/system for determining the cell proliferation rate or celldoubling time of a cell sample, (b) a component/system for selectingand/or retrieving cell samples having a desired cell proliferation rateor cell doubling time, and (c) a component/system for plating theselected samples in a multi-well plate. Similarly, in some embodiments,the present invention provides an automated data-driven batching systemthat comprises: (a) a component/system for determining the cellproliferation rate or cell doubling time of a cell sample, (b) acomponent/system for cryopreserving a cell sample, (c) acomponent/system for selecting and/or retrieving cryopreserved cellsamples having a desired cell proliferation rate or cell doubling time,(d) a component/system for thawing the selected cryopreserved cellsamples, and (e) a component/system for plating the selected samples ina multi-well plate. In some such embodiments, the component/system ofthe data-driven batching system used to determine the cell proliferationrate or cell doubling time of a cell sample may comprise an automatedcell imager (such as a Celigo imager) or other automated device that canbe used to measure cell confluency or cell numbers, or some otherparameter from which cell proliferation rate or cell doubling time canbe calculated (such as a confluency checking unit). In some suchembodiments, the component/system for cryopreserving the cell sample maycomprise a system for robotically transferring cell samples intocryopreservation tubes (such as barcoded cryopreservation tubes) and maycomprise a 80° C. freezer. In some such embodiments, thecomponent/system for selecting cell samples having a desired cellproliferation rate or cell doubling time may comprise a computer systemprogrammed with desired selection criteria, and/or may comprise anautomated sample access system, such as a −80° C. Sample Access Manageror “SAM” (Hamilton Storage Technologies). Such an automated sampleaccess system may comprise an inventory database that allows forflexible recall and downstream process batching of cell samples, forexample based on one or more factors such as cell proliferation rate,cell doubling time, or any other desired property or characteristic.

In some embodiments such data-driven batching systems form part of alarger automated system, such as the larger automated systems describedherein for the generation of iPSCs and/or differentiated cells. Forexample, in one embodiment the present invention provides an automatedsystem for generating and isolating iPSCs, comprising: (a) a somaticcell plating unit for placing somatic cells on a plate; (b) adata-driven batching system used to select somatic cell samples forreprogramming, and (c) an induction unit for automated reprogramming ofthe somatic cells by contacting the somatic cells on the somatic cellplating unit with reprogramming factors to produce iPSCs. In one suchembodiment, the system further comprises a sorting unit for selectivelysorting and isolating the iPSCs produced by the induction unit, e.g., byidentifying iPSC specific markers, including, e.g., surface markers onthe cells. In one illustrative example, the somatic cells arefibroblasts. Similarly, in another embodiment the present inventionprovides an automated system for generating and isolating differentiatedadult cells from stem cells, e.g., iPSCs, embryonic stem (ES) cells ormesenchymal stem (MS) cells, comprising: (a) a stem cell plating unitfor placing stem cells on a plate; (b) a data-driven batching systemused to select stem cell samples for subsequent differentiation and (c)an induction unit for automated differentiation of stem cells, forexample by contacting the cells on the with one or more differentiationfactors to produce differentiated cells. In one such embodiment, thesystem further comprises a sorting unit for selectively sorting andisolating the differentiated cells produced by the induction unit.

As described above, it is a discovery of the present invention that theefficiency of cellular reprograming can be improved, and variability inreprogramming efficiency reduced, when cells are grown in low serum orserum free medium for a certain amount of time prior to, and optionallyalso during, contacting cells with reprogramming factors. While theApplicants, like others, found that increased growth rates werebeneficial for reprogramming, Applicants' findings suggested thatswitching cells to low serum medium at the time of, or soon after,reprogramming may shock the cells and have a detrimental effect onreprogramming success. Applicants found that such effects could bemitigated by allowing the cells to adjust to low serum conditions for acertain amount of time prior to reprogramming. Accordingly, in oneembodiment the present invention provides a method for the generation ofinduced pluripotent stem cells (iPSCs) from differentiated adult somaticcells, the method comprising: (a) obtaining adult somatic cells, (b)culturing the adult somatic cells in low serum medium days, and (c)subsequently contacting the adult somatic cells with one or morereprogramming factors, in order to produce iPSCs. In some suchembodiments the population of adult somatic cells obtained in step (a)had previously been frozen. In some such embodiments the population ofadult somatic cells had been thawed in low serum medium. In some suchembodiments the adult somatic cells are fibroblasts. In some suchembodiments the adult somatic cells are cultured in low serum medium formore than 1 day prior to contacting the adult somatic cells with one ormore reprogramming factors. In some such embodiments the adult somaticcells are cultured in low serum medium for more than 2 days prior tocontacting the adult somatic cells with one or more reprogrammingfactors. In some such embodiments the adult somatic cells are culturedin low serum medium for more than 3 days prior to contacting the adultsomatic cells with one or more reprogramming factors. In some suchembodiments the adult somatic cells are cultured in low serum medium formore than 4 days prior to contacting the adult somatic cells with one ormore reprogramming factors. In some such embodiments the adult somaticcells are cultured in low serum medium for more than 5 days prior tocontacting the adult somatic cells with one or more reprogrammingfactors. In some such embodiments the adult somatic cells are culturedin low serum medium for more than 6 days prior to contacting the adultsomatic cells with one or more reprogramming factors. In some suchembodiments the adult somatic cells are cultured in low serum medium formore than 7 days prior to contacting the adult somatic cells with one ormore reprogramming factors. In some such embodiments the adult somaticcells are cultured in low serum medium for 5-7 days prior to contactingthe adult somatic cells with one or more reprogramming factors. In somesuch embodiments the adult somatic cells are cultured in low serummedium for 4-8 days prior to contacting the adult somatic cells with oneor more reprogramming factors. In some such embodiments the adultsomatic cells are cultured in low serum medium for 3-9 days prior tocontacting the adult somatic cells with one or more reprogrammingfactors. In some such embodiments the adult somatic cells are culturedin low serum medium for 2-10 days prior to contacting the adult somaticcells with one or more reprogramming factors. In some such embodimentsthe step of contacting the adult somatic cells with one or morereprogramming factors is performed while the adult somatic cells are inlow serum medium. In some such embodiments the low serum mediumcomprises less than 5% serum. In some such embodiments the low serummedium comprises less than 4% serum. In some such embodiments the lowserum medium comprises less than 3% serum. In some such embodiments thelow serum medium comprises less than 2% serum. In some such embodimentsthe low serum medium comprises less than 1% serum. In some suchembodiments the low serum medium is serum free. In some such embodimentsthe low serum medium comprises a serum replacement. Several differentserum replacements are known in the art and any such serum replacementcan be used.

In certain embodiments the present invention also provides cells, suchas somatic cells (e.g., donor-derived fibroblasts), pluripotent stemcells (such as iPSCs), differentiated cells produced from pluripotentstem cells (such as hematopoetic cells, muscle cells, cardiac musclecells, liver cells, cartilage cells, epithelial cells, urinary tractcells, and neuronal cells), and transdifferentiated cells, such as thoseproduced using the methods and systems described herein. The presentinvention also provides “arrays” or “panels” or “banks” comprising suchcells. In some embodiments such cell arrays, panels or banks maycomprise cells derived from multiple different individuals, for examplemultiple different individuals in a “population of interest.” Thepopulation of interest can be any population desired, including, but notlimited to, the world population, the population of a particularcountry, the population of a particular continent, the population of aparticular geographic region, the population of a particular racial orethnic group, a population of a particular age, a population of aparticular sex (male or female), a population having a particulardisease or disorder, a population having a particular mutation, apopulation having a particular genotype, a population having aparticular phenotype, a population having a particular blood type, apopulation having a particular HLA type, a population having aparticular drug response profile, and the like. In some embodiments theindividuals from whom cells are derived are selected in order to berepresentative of the variation in the particular population ofinterest. For example, if the population of interest is the U.S.population, the individuals from whom cells are derived are preferablyselected to be representative of the U.S. population (e.g., in terms ofrace/ethnicity and/or any other desired characteristic), for examplebased on census data or some other suitable criteria. In someembodiments the cell panels comprise isogenic control cells. Forexample, in embodiments where the cell panels contain cells having acertain mutation, the panels may comprise control cells in which thatmutation is not present (for example if it has been corrected) but wherethe cells are otherwise genetically identical. In some embodiments thecells in the cell panels comprise a reporter gene, such as a reportergene that can be used to report expression of a gene or gene productthat is or may be involved in a disease, or is in a pathway that is ormay be involved in a disease. In some embodiments the cell panelscomprise both sporadic and familial lines. For example, in the case of apanel containing samples from subjects having a certain disease, thepanel may comprise samples from subjects in which that disease arose asthe result of a sporadic mutation, as well as samples from subjects inwhich that disease was inherited. In some embodiments, the cell panelscomprise 3 or more cell lines/clones from each subject, in order toprovide replicates of samples from each subject. In some embodiments thepresent invention provides populations of stem cells (such as iPSCs) ordifferentiated cells wherein the populations of cells are derived fromat least 96, or at least 384, or at least 1596 different individualsfrom the population of interest. In preferred embodiments cells fromdifferent individuals are provided in separate vessels, such as separatewells of a 96-well, 384-well, or 1596-well microtiter plate.

The cells, arrays, panels and banks of the invention may be useful in avariety of different applications, for example in studying ordetermining the efficacy, toxicity, teratogenicity or safety of, one ormore candidate drugs on cells of different individuals in a populationof interest. As such the cell panels of the invention can be used toperform “clinical trials in a dish.”

In some embodiments the present invention also provides gene sets andprobe sets that may be useful for detection of iPSCs or differentiatedcells, such as those made using the automated systems of the presentinvention. Such gene and probe sets can be used in as part of one of theautomated systems of the invention or in other applications, as desired.

In one embodiment the present invention provides a “Pluri25” gene/probeset comprising the following genes, or probes for detecting expressionof the following genes: retroviral tOct4, retroviral tSox2, retroviraltKlf4, retroviral tC-Myc, Sendai tOct4, Sendai tSox2, Sendai tKlf4,Sendai tC-Myc, Sendai vector (SeV), POU5F1 (Oct4), SOX2, KLF4, MYC,LIN28, NANOG, ZFP42, SOX17, AFP, NR2F2, ANPEP (CD13), ACTB, POLR2A,ALAS1, SRY and XIST.

In another embodiment the present invention provides a gene/probe setfor detection of iPSCs comprising the following genes, or probes fordetecting expression of the following genes: Sendai tOct4, Sendai tSox2,Sendai tKlf4, Sendai tC-Myc, Sendai vector (SeV), POU5F1 (Oct4), SOX2,KLF4, MYC, LIN28, NANOG, ZFP42, SOX17, AFP, NR2F2, ANPEP (CD13), ACTB,POLR2A, ALAS1, SRY and XIST.

In another embodiment the present invention provides a gene/probe setfor detection of iPSCs comprising the following genes, or probes fordetecting expression of the following genes: retroviral tOct4,retroviral tSox2, retroviral tKlf4, retroviral tC-Myc, POU5F1 (Oct4),SOX2, KLF4, MYC, LIN28, NANOG, ZFP42, SOX17, AFP, NR2F2, ANPEP (CD13),ACTB, POLR2A, ALAS1, SRY and XIST.

In another embodiment the present invention provides a gene/probe setfor detection of iPSCs comprising the following genes, or probes fordetecting expression of the following genes: POU5F1 (Oct4), SOX2, KLF4,MYC, LIN28, NANOG, ZFP42, SOX17, AFP, NR2F2, ANPEP (CD13), ACTB, POLR2A,ALAS1, SRY and XIST.

In another embodiment the present invention provides a gene/probe setreferred to as the “3GLSC100” gene/probe set, which is useful fordetection of iPSCs and which comprises the genes, or probes fordetecting expression of the genes, listed in Table 2 in the DetailedDescription section of the present application.

In some embodiments the present invention also provides gene/probe setsfor detection of cells that have begun to differentiate intocardiomyocytes. One such gene/probe set, referred to as the “cardiac 1”gene/probe set, comprises the following genes, or probes for detectingexpression of the following genes: ACTN1, BMP4, GATA4, GJA1, IRX-4,ISL1, KDR, MEF2A, MEF2C, MESP1, MYH6, MYH7, MYL2, MYL7, NKX2-5, NPPA,PDGFRa, SIRPA, TBX20, TBX5, TNNI3, TNT2, VCAM1, VWF, MIXL1, NANOG, OCT4,SOX17, Brachury T and KCNJ2. Another such gene/probe set, referred to asthe “cardiac 2” gene/probe set, comprises the following genes, or probesfor detecting expression of the following genes: ACTN1, BMP4, GATA4,GJA1, IRX-4, ISL1, KDR, MEF2A, MEF2C, MESP1, MYH6, MYH7, MYL2, MYL7,NKX2-5, NPPA, PDGFRa, SIRPA, TBX20, TBX5, TNNI3, TNT2, VCAM1, VWF,MIXL1, NANOG, OCT4, SOX17, Brachury T, KCNJ2, GAPDH, GUSB, HPRT1, andTBP.

In another embodiment the present invention provides methods forobtaining reprogrammed human fibroblasts from mixed cell populations(for example using the automated methods described herein) by isolatingcells that are CD13-negative, SSEA4-positive and Tra-1-60-positive,wherein the CD13-negative, SSEA4-positive and Tra-1-60-positive cellsare reprogrammed human fibroblasts. In some embodiments such methodsutilize fluorescence activated cell sorting (FACS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Steps for acquiring a fibroblast cell bank.

FIG. 2. Steps for obtaining a stem cell array from a fibroblast bank.

FIG. 3. Flowchart showing steps in a system for producing iPSCs.

FIGS. 4A-4C. Examples of a flow of patient samples through multi-welltissue culture plates during an automated reprogramming process.

FIGS. 5A-5C. Examples of an equipment configuration to accomplish theworkflow.

FIGS. 6A-6C. Automated biopsy outgrowth tracking system. In FIG. 6A,biopsies or discarded tissue are plated in multiple wells of a 6-welldish and maintained by an automated system that feeds, images, passages,and freezes fibroblast outgrowths. Examples of the image analysisinterface are shown for a typical sample. FIG. 6B: Cell numbers areextrapolated from confluence measurements based on linear regressionfrom a standard curve generated independently. FIGS. 6C and 6D: Anexample of cell counts for a typical biopsy outgrowth maintained on anautomated system provided by the invention. Extrapolated cell numbersper patient sample are plotted for each well independently (FIG. 6C)allowing calculation of total output from the sample (FIG. 6D).

FIGS. 7A-7D. FACS analyses and graphs showing automated iPSCreprogramming. Expression levels of pluripotent surface markers onreprogrammed human fibroblasts were followed over a 3 week period toobserve reprogramming kinetics and determine optimal time points atwhich to isolate defined cell populations. FIG. 7A FACS gating schemeused for analysis. FIG. 7B: A substantial proportion of cellsco-expressing traditional pluripotency surface markers SSEA4 & TRA-1-60retain the fibroblast marker CD13 at all time points duringreprogramming using viral vectors to introduce reprogramming factorssuch as Oct4, Sox2, Klf4 and c-Myc. Box plots indicating aggregated datafrom 131 experiments (Retrovirus, n=66, Sendai virus, n=65) are shown.While Sendai mediated reprogramming produces more SSEA4/TRA-1-60 doublepositive cells, (FIG. 7C) there is a delay in elimination of CD13 fromthe surface. (FIG. 7D) Example staining pattern of a patient cell linereprogrammed using Sendai/Cytotune system on an automated systemprovided by the invention. At both 7 and 13 days post infection (dpi),more than half of SSEA4/TRA-1-60 double positive cells have lost CD13.Additionally, at both time points assayed, CD13 negative/Nanog positivecells are present in this fraction, suggesting these can be isolated bynegative selection against CD13.

FIGS. 8A-8C. FACs pre-sort analyses and a part of the automated systemto demonstrate enrichment and clone selection of iPSCs. FIG. 8A showsNon-reprogrammed cell populations can be depleted from cultures of iPSCsby negative selection by a fibroblast marker. In the example,fibroblasts are efficiently removed from the culture containing 2%established iPSCs leaving TRA-1-60 positive iPSCs untouched.

FIG. 8B shows a Miltenyi MultiMACS system integrated into Hamiltonliquid handler that can sort 24 samples in parallel. FIG. 8C is anillustration of the iPSC-enriched fraction from the anti-fibroblastmagnetic negative selection step that is plated on 96-well imagingplates at limiting dilution. These plates are screened using live-cellstaining for the pluripotency surface marker TRA-1-60 or TRA-1-81. Wellswith TRA-1-60 positive iPSCs are identified by automated image analysisusing the Celigo software capable of single colony confirmation. Wellsthat meet both criteria of containing a single colony that is positivefor the surface marker are selected for passaging, expansion, and QC.

FIGS. 9A-9B. Illustration for the scorecard assays described herein. Thefirst stage of the quality control screen uses a panel of pluripotencydifferentiation and transgene markers to choose an initial set of threeclones. (FIG. 9A) Transcript counts after normalization to HK geneexpression for two human ESC lines, Sendai positive control, fibroblastnegative control, and iPSC lines derived by FACS sorting assayed atpassage 5 and 10. All assays are run relative to a panel of normal humanESC and iPSC lines maintained under similar conditions. (FIG. 9B) Secondstage of a quality control screen, which uses an additional 83 germlayer/lineage markers to monitor differentiation capability in embryoidbody assays. Single EBs are generated and pooled to collect RNA forexpression analysis of germ layer markers in the embryoid body scorecardassay. Shown is a cluster dendrogram analysis of gene expression in EBscollected from nine different embryonic stem cells lines. Afternormalization, data generated from direct lysis of six EBs comparesfavorably to data generated from total RNA extracted and purified fromEBs prepared from bulk culture.

FIGS. 10A-10B. High throughput karyotyping of iPSCs based on NanostringnCounter assays for CNVs. (FIG. 10A) is an example of the nCounterKaryotype assay on BC1 iPSCs; (FIG. 10B) is an example of the nCounterKaryotype assay on 1016 fibroblasts with partial gain and loss ofchromosome arms. Comparison to Affymetrix SNP 6.0 chip datademonstrating copy number gains on a portion of the q arm of Chr1 (toptrack, 1q21.2-1q43) and loss of part of the long arm of Chr6 (bottomtrack, 6q16.3-6q26).

FIGS. 11A-11E. Enhanced derivation and maintenance of virallyreprogrammed fibroblasts using Fluorescence Activated Cell Sorting.(FIG. 11A) CD13NEGSSEA4POSTra-1-60NEG and CD13NEGSSEA4POSTra-1-60POSpopulations from the manually derived 1018 clone were sorted onto MEFfeeder layers and expanded for 20 days prior to reanalysis by flowcytometry to assess retention of sorted surface markers. dpi=days postinfection. dps=days post sort. (FIG. 11B) CD13NEGSSEA4POS andCD13NEGSSEA4POSTra-1-60POS populations were sorted onto MEF layers atseven days post infection and imaged at 3 and 17 dps to assess colonyformation. (FIG. 11C) Colony counts arising from the sorted cellpopulations shown in Panel B at 17 dps (25 dpi). (FIG. 11D) Gatingstructure used in the analysis of CD13POS cells present within theSSEA4POSTra-1-60POS population at 7 dpi. (FIG. 11E) Fluorescencemicroscopy demonstrating NANOG expression in CD13POS cell at 7 dpi. 40×magnification. CD13 shown in red. Nanog in shown Green. Valuesdesignated % T indicates proportion of total cells within the culturepositive for the indicated combinations of surface markers. Valueswithout T designation indicate the proportion of CD13NEGSSEA4POS cellsthat are Tra-1-60POS or Tra-1-60NEG in Panel A and D.

FIGS. 12A-12D. Fibroblasts undergoing viral reprogramming exhibitcharacteristic expression levels of surface markers at early time pointspost infection. (FIG. 12A) Foreskin (0825) and adult dermal fibroblast(1018 and 1023) lines underwent four factor retroviral reprogramming andwere analyzed by flow cytometry for the emergence of theCD13^(NEG)SSEA4^(POS)Tra-1-60^(POS) population at seven day intervalspost infection. Values designated % T indicates proportion of totalcells within the culture positive for the indicated combinations ofsurface markers. Values without T designation indicate the proportion ofcells positive within the parent gate. (FIG. 12B) Gating structure usedto sort the CD13^(NEG)SSEA4^(POS)Tra-1-60^(POS) populations for all celllines derived in this study. Live cell are first defined using forward(FSC) and Side (SSC) light scattering properties. TheCD13^(NEG)SSEA4^(POS) population is then selected from the live cellgate (blue cells). The highest Tra-1-60^(POS) expressing cells are thenselected from the CD13^(NEG)SSEA4^(POS) population (Green cells) andsorted for expansion and characterization. (FIG. 12C) Comparison ofSSEA4^(POS)Tra-1-60^(POS) populations present in Retro (R) or Sendai (S)viral infected fibroblast cultures during first two weeks ofprogramming. (FIG. 12D) Comparison of CD13^(POS) cells present withinthe SSEA4^(POS)Tra-1-60^(POS) populations during first two weeks ofprogramming following Retro (R) or Sendai (S) infection. Dpi 1-7:(R)n=29, (S) n=21. Dpi 8-14: (R) n=32, (S) n=46. Total n=228. Statisticalsignificance was assessed via Student's t-Test. * p<=0.05, ** p<=0.001,*** p<=0.001, ****p<=0.0001.

FIGS. 13A-13C. Fluorescence Activated Cell Sorting generates higherquality independent clones than manual derivation. Modified pluripotentscorecard assay was performed on manually and FACS derived clones todemonstrate (FIG. 13A) activation of endogenous gene expression and(FIG. 13B) silencing of gene expression and presence of unreprogrammedand transformed fibroblasts CD13^(POS) in manually derived clones. (FIG.13C) Three sorted and three picked lines from patient 1023 were used tocompare the ability of both methods to generate independent clones. 10μof genomic DNA were cut overnight with BglII and submitted to Southernblotting. The HUES line HES2 was used as a positive control forendogenous KLF4/OCT4, and as a negative control for transgeneinsertions. Samples were first blotted for KLF4, then stripped andreblotted for OCT4. Picked clones 1023 C and E are consistent with beingthe same clone by both KLF4 and OCT4 blotting. * indicated the predictedendogenous KLF4/OCT4 bands, and ** indicated a consistent band found inall samples blotted with OCT4.

FIGS. 14A-14E. hIPSC lines derived by Fluorescence Activated CellSorting possess in vitro and in vivo spontaneous differential potential.Embryoid bodies were derived from FACS (FIG. 14A) or manually derivedclones (FIG. 14B) and stained for expression of alpha fetoprotein,smooth muscle actin and beta III tubulin (Tuj1) to demonstratedifferentiation potential in vitro potential. 10× Magnification (FIG.14C) Differentiation potential of the derived lines for expression ofgerm layer genes present in the Lineage scorecard assay. (FIG. 14D)Teratomas from FACS (FIG. 14D) or manually derived (FIG. 14E) clones of1023 fibroblast line indicating in vitro differentiation potential byformation of three germ layers.

FIGS. 15A-15B. Stability of Fluorescence Activated Cell Sorted andManually Derived IPSC Lines. Three individual clones were selected fromforeskin (0819) fibroblasts lines which previously underwent four factorretroviral reprogramming and were derived by either FACS (FIG. 15A,C₁-C₃) or manual (FIG. 15B, C₄-C₆) techniques were analyzed by flowcytometry for pluripotent surface marker expression following expansionon murine embryonic fibroblasts for 12-14 passages. Clones C3 and C6were adapted to Matrigel and mTSER media around passage 11 and expandedfor several passages prior to surface marker analysis by flow cytometryto demonstrate stability following changes in culture conditions. Eventsdisplayed in the 2D scatter plots are “live” cells as defined by forwardand side scatter properties expressing indicated surface markers.

FIGS. 16A-16G. Characterization of Fluorescence Activated Cell Sortedand Manually Derived IPSC Lines by Sendai virus. Immunofluroescencemicroscopy of the 1001.131.01 line demonstrating expression of commonmarkers of pluripotency by FACS or Manually Derived IPSC lines. NuclearTranscription Factors shown in Green, Surface Markers shown in Red,Nucleus stained with DAPI in Blue (FIG. 16A) Nanog/Tra-1-60 (FIG. 16B)Oct4/Tra-1-81 (FIG. 16C) Sox2/SSEA4 (FIG. 16D) Oct4/AlkalinePhosphatase. 10× Magnification (FIG. 16E) Expression of endogenouspluripotent transcription factors (FIG. 16F) Silencing of viraltranscription factors occur by passage 5. (FIG. 16G) Expression levelsof transcription factors common to the indicated germ layers from EBgenerated by the indicated IPSC lines.

FIG. 17. Time Course analysis of retroviral reprogrammed fibroblasts.The 0825 foreskin fibroblast line was analyzed for changes inpluripotent surface marker expression by flow cytometry at −7 dpiintervals following retroviral reprogramming to determine earliest timepoint at which the CD13^(NEG)SSEA4^(POS)Tra-1-60^(POS) populationappears. Values indicate percent of total cells in the cultureexpressing the indicated markers.

FIGS. 18A-18B. Karyotype of FACS (FIG. 18A) and Manually (FIG. 18B)Derived retroviral iPS lines possess a normal karyotype and match theparent fibroblast. Karyotype was assessed using 20 G-banded metaphasecells from each fibroblast and reprogrammed lines at passages indicated.All lines possess a normal karyotype and match the parent fibroblast.Karyotype was assessed using 20 G-banded metaphase cells from eachfibroblast and reprogrammed lines at passages indicated. Fibroblasts andFACS derived lines possess a normal karyotype and match the parentfibroblast. Three out of 20 cells from the manually derived linedisplayed an unbalanced translocation between the short arm ofchromosomes 11 and 22 resulting in trisomy of the short arm ofchromosome 11.

FIGS. 19A-19D. FACS and Manually Derived Sendai iPS lines expresspluripotency markers. FACS (FIG. 19A) or Manually (FIG. 19B) derivedclones were expanded on MEF feeder layers and stained for two commonmarkers of pluripotency: Tra-1-60 and Nanog. 10× Magnification. Alllines show consistent expression of pluripotency markers. (FIG. 19C)qRTPCR showing expression of endogenous gene expression and silencing(FIG. 19D) of retroviral genes.

FIGS. 20A-20G. Automated fibroblast and iPSC production. (FIG. 20A)Schematic of workflow through automation system from donor biopsycollection through to iPS expansion and freezing. (FIG. 20B) Image ofsystem for automated fibroblast production consisting of a liquidhandling device, imager, centrifuge and capper/decaper contained in abiosafety cabinet, connected to an automated incubator and managed bysystem control software. (FIG. 20C) Phase contrast image ofrepresentative fibroblast outgrowth from a biopsy. (10×) (FIG. 20D)Fibroblast biopsy outgrowth (i) as visualized following automatedimaging. Confluence measurements (ii) and Hoechst stained nuclei (iii)are compared against each other to generate a regression model (iv) forcalculating count values from unstained samples with confluencemeasurements. (FIG. 20E) Histogram of fibroblast doubling timescalculated from confluence scans of fibroblasts during expansion. (FIG.20F) Scatterplot of doubling time vs. age of donor. (FIG. 20G)Fibroblast doubling times from fibroblasts thawed and recovered forreprogramming.

FIGS. 21A-21I. Automated reprogramming. (FIG. 21A) Experimental schemefor automated fibroblast thawing and reprogramming. (FIG. 21B) Image ofrobotic system for automated fibroblast thawing and mRNA transfections.(FIG. 21C) Timecourse of mRNA transfection showing development ofcolonies over 22 days. (FIG. 21D) Image-based identification andcounting of TRA-1-60 positive colonies to determine reprogrammingefficiency. (FIG. 21E) FACS analysis of reprogrammed cultures fromautomated mRNA transfection demonstrating that higher proportion ofcells after reprogramming by mRNA stain double positive for thepluripotency markers TRA-1-60/SSEA4 and lack of the fibroblast surfacemarker CD13. (FIG. 21F, FIG. 21G, FIG. 21H and FIG. 21I) Effect plots ofpoison regression analysis of factors that contribute to reprogrammingsuccess. Gray area and bars indicate confidence intervals.

FIGS. 22A-22G. Automated iPSC purification and arraying. (FIG. 22A)Schematic illustration of bulk method of unreprogrammed fibroblast celldepletion from reprogramming 24 well plates, consolidation and freezing.(FIG. 22B) Image of integrated magnetic bead selection robot. (FIG. 22C)Representative images of 96-well for bulk sorted cells from first daypost sorting (1 dps) to 9 days post sorting (9dps), and TRA-1-60expression pattern captured by automated imaging. (FIG. 22D) Example ofa 96-well plate of sorted iPSCs derived from mRNA mediatedreprogramming. Three samples are represented on the plate in three-pointtwo fold serial dilution. (FIG. 22E) Representative images forretrospective identification of clonal lines derived from sorted cellsfrom a single cell identified on the second day post sorting (2dps) toits colony at 10 days post sorting (10 dps), and TRA-1-60 expressionpattern captured by Celigo Tumorsphere application. (FIG. 22F) FACSanalysis for TRA-1-60/SSEA4/CD13 on sorted cells after consolidation andprior to freezing. (FIG. 22G) Histogram of doubling time for iPSCsduring growth after sorting before freezing. Frequency bins are 6 hrs.Median doubling time was 42 hr, n=826 samples.

FIGS. 23A-23B. Gene expression analysis of sorted cells. (FIG. 23A)Boxplot of the pluripotency scores for reference hESC lines, iPSC lines,and fibroblast lines. (FIG. 23B) Boxplot of differentiation scores forthe three categories of cell lines.

FIGS. 24A-24F. Automated parallel iPSC culture. (FIG. 24A) Image of96-well freezing and passaging robot. (FIG. 24B) Bright field images ofiPS cells in the same well in a 96W plate recovering after automatedthawing method. Confluence was monitored over 5 days. (FIG. 24C)Correlation of confluence data from the Celigo prior to cryotube freezeand post-thaw. Both the freezing and thawing of cryotubes were performedon the integrated automated system. (FIG. 24D) FACS analysis ofTRA-1-60/SSEA4 double positive population before and after automatedpassaging for control hESCs and iPSCs derived on the system. (FIG. 24E)FACS analysis of iPSCs before freezing and recovered from thawing usingautomated methods. (FIG. 24F) Example growth rates of a roboticallypassaged iPSC plate over 3 days culture.

FIGS. 25A-25F. Automated Embryoid body assay. (FIG. 25A) Boxplot of thedifferentiation propensities (EC=ectoderm, ME=mesoderm, EN=endoderm) forthe 10 hESC reference lines with segments showing the averagedifferentiation propensities observed for iPSC lines derived using threedifferent methods. (FIG. 25B) Representative image of the Greiner 96well v-bottom plate with EBs is shown after passage to form EBs byautomation. The EBs were generated from iPSC lines ubiquotouslyexpressing GFP. (FIG. 25C) Image of EBs by stereomicroscopy. (FIG. 25D,FIG. 25E and FIG. 25F) Correlation of differentiation propensity for allsamples generated using the reference lines used in this study withpreviously published scorecard reference data (Bock et al., 2011).

FIG. 26. Reduced variation in robotically derived iPSCs. Varianceanalysis of scorecard gene expression in EBs showing comparisons ofstandard deviation of gene expression values among samples derived onand off the automated system. Lines produced by the automated processshowed significantly less variation in EB gene expression compared tolines produced by manual methods and later introduced onto the system (pvalue=7.08E-12, Wilcoxen signed rank test). The comparison betweenmanually derived lines and lines reprogrammed on the automation but withmanual colony picking was marginally significantly different (pvalue=0.023) *=p<0.05, ***=p<0.001).

FIGS. 27A-27B. (FIG. 27A) Mycoplasma detection of samples from in-houseautomated luminescence assay. Marginal values were confirmed negativewith PCR validation. No mycoplasma positive samples have been generatedin house during biopsy collection and outgrowth expansion on theautomated systems. (FIG. 27B) Representative traces of fibroblastskaryotyped using the Nanostring Karyotype assay with representativetraces of normal diploid fibroblasts (a) 46, XX, (b) 46, XY and ananeuploid fibroblast showing a loss of one X chromosome (c) 45, X.

FIGS. 28A-28E. Comparisons of automated reprogramming by mRNA andSendai. (FIG. 28A) Pluripotency staining of mRNA derived lines andexample of a phase contrast image of iPSC produced by mRNA transfection.Scale bars are 50 μm. (FIG. 28B) Well image of Sendai reprogrammingafter 20 days showing colonies and TRA-1-60 live stain of the same wellin the bottom panel. Only a subset of colonies stain positive for thepluripotency marker. (FIG. 28C) Pluripotency marker staining ofestablished cell lines from automated reprogramming by Sendai virus.Scale bars are 50 μm. (FIG. 28D) FACS analysis of reprogrammed culturesfrom automated mRNA transfection demonstrating that higher proportion ofcells after reprogramming by mRNA stain double positive for thepluripotency markers TRA-1-60/SSEA4 and lack of the fibroblast surfacemarker CD13. (FIG. 28E) Variation of gene expression of the germ layerand pluripotency markers shown in (FIG. 28D) for BJ fibroblastsreprogrammed on the automated system by mRNA transfection or Sendaiinfection and isolated by manual colony picking. Means are notsignificantly different. N=33.

FIGS. 29A-29C. (FIG. 29A) iPSC:fibroblast (1:20 to 1:100) were mixed, 5%of total cells before and after magnetic bead negative selection usinganti-fibroblast microbeads were analyzed using FACS. Representative datais shown pre and post-sort of a mixture of iPSCs and adult humanfibroblasts at a 1:50 ratio. (FIG. 29B) Representative FACS result for5% of total cells before and after magnetic bead negative selectionusing anti-fibroblast microbeads. (FIG. 29C) Examples of clonal linesderived by automated sorting method.

FIGS. 30A-30C. (FIG. 30A) Pluri25 scorecard assay values for T scoresand gene expression means. (FIG. 30B) Immunofluoresence result for Nanogexpression in consolidated cells in 96 well format. (FIG. 30C) Exampleof overgrown well demonstrating spontaneous differentiation detectibleby the Pluri25 scorecard assays.

FIGS. 31A-31H. (FIG. 31A) Schematic of thawing and passaging and platereplication in 96 well format. (FIG. 31B) Percent coefficient ofvariation was calculated for 3 plates from the replication passages of arepresentative cell line using data from confluence scans from 3different time points. (FIG. 31C) Immunostaining of iPS lines derived onthe robotic platform as well as control hES cell lines showing thepresence of POU5FI and TRA_1-81; SSEA4 and NANOG, and SOX2 and TRA-1-81.(FIG. 31D) FACS analysis of TRA-1-60/SSEA4 double positive populationbefore and after automated passage 1:3 for control hESC lines and iPSClines derived on the system. (FIG. 31E) Detection of an aneuploid linein 4 of 38 independent iPSC lines tested using the Nanostringkaryotyping assay. (FIG. 31F, FIG. 31G and FIG. 32H) Nanostring identitytest data comparing Fibroblast lines to iPSCs derived by the automatedprocess.

FIGS. 32A-32B. (FIG. 32A) Pluripotency marker analysis of referencelines used for lineage scorecard analysis after adaptation to serum-freemedia used to grow iPSCs on the system. EBs were generated from theselines by the automated system under identical conditions to those usedto generate EBs from iPSCs generated on the system. (FIG. 32B) Scorecardanalysis of EBs generated from reference HUES lines under automatedconditions.

FIGS. 33A-33B. (FIG. 33A) Comparisons of standard deviation of geneexpression values for different types of cell lines considering only asingle replicate per sample. (FIG. 33B) Significance test: −log10(p-value) using the Wilcoxen signed rank test that tests the nullhypothesis that the median of these paired distributions is the same(non-parametric test).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides various improved systems and methods forobtaining, generating, culturing, and handling cells, such as stem cells(including induced pluripotent stem cells or iPSCs) and differentiatedcells, as well as cells and cell panels produced using such systems andmethods, and uses of such cells and cell panels. The present inventionbuilds upon, and provides certain improvements over, the systems andmethods described previously in international patent applicationPCT/US2012/067417 (published on Jun. 6, 2013 with publication numberWO/2013/082509) and U.S. patent application Ser. No. 13/691,258(published on Dec. 26, 2013 with publication number 2013-0345094), thecontents of each of which are hereby incorporated by reference in theirentireties for those jurisdictions that permit incorporation byreference. Building on the work described in these prior patentapplications, the Applicants have now developed certain improved methodsand systems that can significantly reduce variability in obtaining,generating, culturing, and handling a variety of cell types, includingdifferentiated cells and stem cells. Such improved systems and methodsare particularly advantageous for automated and/or high-throughputapplications—greatly facilitating the ability to work with large numbersof cells in a parallel manner.

Several of the major embodiments of the present invention are describedin the above “Summary of the Invention” section of this application, aswell as in the Examples, Figures, and Claims sections of this patentapplication. In order to avoid unnecessary duplication, such embodimentsmay not be described in full in this Detailed Description section.Rather this Detailed Description provides certain additional informationregarding the invention, which is intended to be read together and inconjunction with the Summary of the Invention section of the applicationand all other sections of this patent application. Furthermore, thevarious embodiments described in each section of this patent applicationare intended to be combined in various ways, as will be apparent tothose of skill in the art.

As used herein “adult” means post-fetal, i.e., an organism from theneonate stage through the end of life, and includes, for example, cellsobtained from delivered placenta tissue, amniotic fluid and/or cordblood.

As used herein, the term “adult differentiated cell” encompasses a widerange of differentiated cell types obtained from an adult organism, thatare amenable to producing iPSCs using the instantly described automationsystem. Preferably, the adult differentiated cell is a “fibroblast.”Fibroblasts, also referred to as “fibrocytes” in their less active form,are derived from mesenchyme. Their function includes secreting theprecursors of extracellular matrix components including, e.g., collagen.Histologically, fibroblasts are highly branched cells, but fibrocytesare generally smaller and are often described as spindle-shaped.Fibroblasts and fibrocytes derived from any tissue may be employed as astarting material for the automated workflow system on the invention.

As used herein, the term, “induced pluripotent stem cells” or, iPSCs,means that the stem cells are produced from differentiated adult cellsthat have been induced or changed, i.e., reprogrammed into cells capableof differentiating into tissues of all three germ or dermal layers:mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer tocells as they are found in nature.

Mammalian “somatic cells” useful in the present invention include, byway of example, adult stem cells, sertoli cells, endothelial cells,granulosa epithelial cells, neurons, pancreatic islet cells, epidermalcells, epithelial cells, hepatocytes, hair follicle cells,keratinocytes, hematopoietic cells, melanocytes, chondrocytes,lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes,mononuclear cells, fibroblasts, cardiac muscle cells, other known musclecells, and generally any live somatic cells. In particular embodiments,fibroblasts are used. The term somatic cell, as used herein, is alsointended to include adult stem cells. An adult stem cell is a cell thatis capable of giving rise to all cell types of a particular tissue.Exemplary adult stem cells include hematopoietic stem cells, neural stemcells, and mesenchymal stem cells.

The term “totipotency” refers to a cell with a developmental potentialto make all of the cells in the adult body as well as theextra-embryonic tissues, including the placenta. The fertilized egg(zygote) is totipotent, as are the cells (blastomeres) of the morula (upto the 16-cell stage following fertilization).

The term “pluripotent” as used herein refers to a cell with thedevelopmental potential, under different conditions, to differentiate tocell types characteristic of all three germ cell layers, i.e., endoderm(e.g., gut tissue), mesoderm (including blood, muscle, and vessels), andectoderm (such as skin and nerve). A pluripotent cell has a lowerdevelopmental potential than a totipotent cell. The ability of a cell todifferentiate to all three germ layers can be determined using, forexample, a nude mouse teratoma formation assay. In some embodiments,pluripotency can also evidenced by the expression of embryonic stem (ES)cell markers, although the preferred test for pluripotency of a cell orpopulation of cells generated using the compositions and methodsdescribed herein is the demonstration that a cell has the developmentalpotential to differentiate into cells of each of the three germ layers.In some embodiments, a pluripotent cell is termed an “undifferentiatedcell.” Accordingly, the terms “pluripotency” or a “pluripotent state” asused herein refer to the developmental potential of a cell that providesthe ability for the cell to differentiate into all three embryonic germlayers (endoderm, mesoderm and ectoderm). Those of skill in the art areaware of the embryonic germ layer or lineage that gives rise to a givencell type. A cell in a pluripotent state typically has the potential todivide in vitro for a long period of time, e.g., greater than one yearor more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that has the developmental potential to differentiateinto cells of one or more germ layers, but not all three. Thus, amultipotent cell can also be termed a “partially differentiated cell.”Multipotent cells are well known in the art, and examples of multipotentcells include adult stem cells, such as for example, hematopoietic stemcells and neural stem cells. “Multipotent” indicates that a cell mayform many types of cells in a given lineage, but not cells of otherlineages. For example, a multipotent hematopoietic cell can form themany different types of blood cells (red, white, platelets, etc.), butit cannot form neurons. Accordingly, the term “multipotency” refers to astate of a cell with a degree of developmental potential that is lessthan totipotent and pluripotent.

The terms “stem cell” or “undifferentiated cell” as used herein, referto a cell in an undifferentiated or partially differentiated state thathas the property of self-renewal and has the developmental potential todifferentiate into multiple cell types, without a specific impliedmeaning regarding developmental potential (i.e., totipotent,pluripotent, multipotent, etc.). A stem cell is capable of proliferationand giving rise to more such stem cells while maintaining itsdevelopmental potential. In theory, self-renewal can occur by either oftwo major mechanisms. Stem cells can divide asymmetrically, which isknown as obligatory asymmetrical differentiation, with one daughter cellretaining the developmental potential of the parent stem cell and theother daughter cell expressing some distinct other specific function,phenotype and/or developmental potential from the parent cell. Thedaughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. A differentiated cell may derive from amultipotent cell, which itself is derived from a multipotent cell, andso on. While each of these multipotent cells may be considered stemcells, the range of cell types each such stem cell can give rise to,i.e., their developmental potential, can vary considerably.Alternatively, some of the stem cells in a population can dividesymmetrically into two stem cells, known as stochastic differentiation,thus maintaining some stem cells in the population as a whole, whileother cells in the population give rise to differentiated progeny only.Accordingly, the term “stem cell” refers to any subset of cells thathave the developmental potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retain the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In some embodiments, the termstem cell refers generally to a naturally occurring parent cell whosedescendants (progeny cells) specialize, often in different directions,by differentiation, e.g., by acquiring completely individual characters,as occurs in progressive diversification of embryonic cells and tissues.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. Cells thatbegin as stem cells might proceed toward a differentiated phenotype, butthen can be induced to “reverse” and re-express the stem cell phenotype,a term often referred to as “dedifferentiation” or “reprogramming” or“retrodifferentiation” by persons of ordinary skill in the art.

The term “embryonic stem cell” as used herein refers to naturallyoccurring pluripotent stem cells of the inner cell mass of the embryonicblastocyst (see, for e.g., U.S. Pat. Nos. 5,843,780; 6,200,806;7,029,913; 7,584,479, which are incorporated herein by reference). Suchcells can similarly be obtained from the inner cell mass of blastocystsderived from somatic cell nuclear transfer (see, for example, U.S. Pat.Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein byreference). Embryonic stem cells are pluripotent and give rise duringdevelopment to all derivatives of the three primary germ layers:ectoderm, endoderm and mesoderm. In other words, they can develop intoeach of the more than 200 cell types of the adult body when givensufficient and necessary stimulation for a specific cell type. They donot contribute to the extra-embryonic membranes or the placenta, i.e.,are not totipotent.

As used herein, the distinguishing characteristics of an embryonic stemcell define an “embryonic stem cell phenotype.” Accordingly, a cell hasthe phenotype of an embryonic stem cell if it possesses one or more ofthe unique characteristics of an embryonic stem cell, such that thatcell can be distinguished from other cells not having the embryonic stemcell phenotype. Exemplary distinguishing embryonic stem cell phenotypecharacteristics include, without limitation, expression of specificcell-surface or intracellular markers, including protein and microRNAs,gene expression profiles, methylation profiles, deacetylation profiles,proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like. In someembodiments, the determination of whether a cell has an “embryonic stemcell phenotype” is made by comparing one or more characteristics of thecell to one or more characteristics of an embryonic stem cell linecultured within the same laboratory.

The term “somatic stem cell” is used herein to refer to any pluripotentor multipotent stem cell derived from non-embryonic tissue, includingfetal, juvenile, and adult tissue. Natural somatic stem cells have beenisolated from a wide variety of adult tissues including blood, bonemarrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle,and cardiac muscle. Each of these somatic stem cells can becharacterized based on gene expression, factor responsiveness, andmorphology in culture. Exemplary naturally occurring somatic stem cellsinclude, but are not limited to, neural stem cells, neural crest stemcells, mesenchymal stem cells, hematopoietic stem cells, and pancreaticstem cells. In some aspects described herein, a “somatic pluripotentcell” refers to a somatic cell, or a progeny cell of the somatic cell,that has had its developmental potential altered, i.e., increased, tothat of a pluripotent state by contacting with, or the introduction of,one or more reprogramming factors using the compositions and methodsdescribed herein.

The term “progenitor cell” is used herein to refer to cells that havegreater developmental potential, i.e., a cellular phenotype that is moreprimitive (e.g., is at an earlier step along a developmental pathway orprogression) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells have significant or very highproliferative potential. Progenitor cells can give rise to multipledistinct cells having lower developmental potential, i.e.,differentiated cell types, or to a single differentiated cell type,depending on the developmental pathway and on the environment in whichthe cells develop and differentiate.

As used herein, the term “somatic cell” refers to any cell other than agerm cell, a cell present in or obtained from a pre-implantation embryo,or a cell resulting from proliferation of such a cell in vitro. Statedanother way, a somatic cell refers to any cell forming the body of anorganism, as opposed to a germline cell. In mammals, germline cells(also known as “gametes”) are the spermatozoa and ova which fuse duringfertilization to produce a cell called a zygote, from which the entiremammalian embryo develops. Every other cell type in the mammalianbody—apart from the sperm and ova, the cells from which they are made(gametocytes) and undifferentiated, pluripotent, embryonic stem cells—isa somatic cell: internal organs, skin, bones, blood, and connectivetissue are all made up of somatic cells. In some embodiments the somaticcell is a “non-embryonic somatic cell,” by which is meant a somatic cellthat is not present in or obtained from an embryo and does not resultfrom proliferation of such a cell in vitro. In some embodiments thesomatic cell is an “adult somatic cell,” by which is meant a cell thatis present in or obtained from an organism other than an embryo or afetus or results from proliferation of such a cell in vitro. Unlessotherwise indicated, the compositions and methods for reprogramming asomatic cell described herein can be performed both in vivo and in vitro(where in vivo is practiced when a somatic cell is present within asubject, and where in vitro is practiced using an isolated somatic cellmaintained in culture).

The term “differentiated cell” encompasses any somatic cell that is not,in its native form, pluripotent, as that term is defined herein. Thus,the term a “differentiated cell” also encompasses cells that arepartially differentiated, such as multipotent cells, or cells that arestable, non-pluripotent partially reprogrammed, or partiallydifferentiated cells, generated using any of the compositions andmethods described herein. In some embodiments, a differentiated cell isa cell that is a stable intermediate cell, such as a non-pluripotent,partially reprogrammed cell. It should be noted that placing manyprimary cells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such differentiated or somaticcells does not render these cells non-differentiated cells (e.g.,undifferentiated cells) or pluripotent cells. The transition of adifferentiated cell (including stable, non-pluripotent partiallyreprogrammed cell intermediates) to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character upon placement in culture. Reprogrammed and, insome embodiments, partially reprogrammed cells, also have thecharacteristic of having the capacity to undergo extended passagingwithout loss of growth potential, relative to parental cells havinglower developmental potential, which generally have capacity for only alimited number of divisions in culture. In some embodiments, the term“differentiated cell” also refers to a cell of a more specialized celltype (i.e., decreased developmental potential) derived from a cell of aless specialized cell type (i.e., increased developmental potential)(e.g., from an undifferentiated cell or a reprogrammed cell) where thecell has undergone a cellular differentiation process.

The term “reprogramming” as used herein refers to a process thatreverses the developmental potential of a cell or population of cells(e.g., a somatic cell). Stated another way, reprogramming refers to aprocess of driving a cell to a state with higher developmentalpotential, i.e., backwards to a less differentiated state. The cell tobe reprogrammed can be either partially or terminally differentiatedprior to reprogramming. In some embodiments of the aspects describedherein, reprogramming encompasses a complete or partial reversion of thedifferentiation state, i.e., an increase in the developmental potentialof a cell, to that of a cell having a pluripotent state. In someembodiments, reprogramming encompasses driving a somatic cell to apluripotent state, such that the cell has the developmental potential ofan embryonic stem cell, i.e., an embryonic stem cell phenotype. In someembodiments, reprogramming also encompasses a partial reversion of thedifferentiation state or a partial increase of the developmentalpotential of a cell, such as a somatic cell or a unipotent cell, to amultipotent state. Reprogramming also encompasses partial reversion ofthe differentiation state of a cell to a state that renders the cellmore susceptible to complete reprogramming to a pluripotent state whensubjected to additional manipulations, such as those described herein.Such manipulations can result in endogenous expression of particulargenes by the cells, or by the progeny of the cells, the expression ofwhich contributes to or maintains the reprogramming. In certainembodiments, reprogramming of a cell using the synthetic, modified RNAsand methods thereof described herein causes the cell to assume amultipotent state (e.g., is a multipotent cell). In some embodiments,reprogramming of a cell (e.g., a somatic cell) using the synthetic,modified RNAs and methods thereof described herein causes the cell toassume a pluripotent-like state or an embryonic stem cell phenotype. Theresulting cells are referred to herein as “reprogrammed cells,” “somaticpluripotent cells,” and “RNA-induced somatic pluripotent cells.” Theterm “partially reprogrammed somatic cell” as referred to herein refersto a cell which has been reprogrammed from a cell with lowerdevelopmental potential by the methods as disclosed herein, such thatthe partially reprogrammed cell has not been completely reprogrammed toa pluripotent state but rather to a non-pluripotent, stable intermediatestate. Such a partially reprogrammed cell can have a developmentalpotential lower that a pluripotent cell, but higher than a multipotentcell, as those terms are defined herein. A partially reprogrammed cellcan, for example, differentiate into one or two of the three germlayers, but cannot differentiate into all three of the germ layers.

The term a “reprogramming factor,” as used herein, refers to adevelopmental potential altering factor, as that term is defined herein,such as a gene, protein, RNA, DNA, or small molecule, the expression ofwhich contributes to the reprogramming of a cell, e.g., a somatic cell,to a less differentiated or undifferentiated state, e.g., to a cell of apluripotent state or partially pluripotent state. A reprogramming factorcan be, for example, transcription factors that can reprogram cells to apluripotent state, such as SOX2, OCT3/4, KLF4, NANOG, LIN-28, c-MYC, andthe like, including as any gene, protein, RNA or small molecule, thatcan substitute for one or more of these in a method of reprogrammingcells in vitro. In some embodiments, exogenous expression of areprogramming factor, using the synthetic modified RNAs and methodsthereof described herein, induces endogenous expression of one or morereprogramming factors, such that exogenous expression of one or morereprogramming factors is no longer required for stable maintenance ofthe cell in the reprogrammed or partially reprogrammed state.“Reprogramming to a pluripotent state in vitro” is used herein to referto in vitro reprogramming methods that do not require and/or do notinclude nuclear or cytoplasmic transfer or cell fusion, e.g., withoocytes, embryos, germ cells, or pluripotent cells. A reprogrammingfactor can also be termed a “de-differentiation factor,” which refers toa developmental potential altering factor, as that term is definedherein, such as a protein or RNA, that induces a cell tode-differentiate to a less differentiated phenotype, that is ade-differentiation factor increases the developmental potential of acell.

As used herein, the term “differentiation factor” refers to adevelopmental potential altering factor, as that term is defined herein,such as a protein, RNA, or small molecule, that induces a cell todifferentiate to a desired cell-type, i.e., a differentiation factorreduces the developmental potential of a cell. In some embodiments, adifferentiation factor can be a cell-type specific polypeptide, howeverthis is not required. Differentiation to a specific cell type canrequire simultaneous and/or successive expression of more than onedifferentiation factor. In some aspects described herein, thedevelopmental potential of a cell or population of cells is firstincreased via reprogramming or partial reprogramming using synthetic,modified RNAs, as described herein, and then the cell or progeny cellsthereof produced by such reprogramming are induced to undergodifferentiation by contacting with, or introducing, one or moresynthetic, modified RNAs encoding differentiation factors, such that thecell or progeny cells thereof have decreased developmental potential.

In the context of cell ontogeny, the term “differentiate”, or“differentiating” is a relative term that refers to a developmentalprocess by which a cell has progressed further down a developmentalpathway than its immediate precursor cell. Thus in some embodiments, areprogrammed cell as the term is defined herein, can differentiate to alineage-restricted precursor cell (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a tissue specific precursor, forexample, a cardiomyocyte precursor), and then to an end-stagedifferentiated cell, which plays a characteristic role in a certaintissue type, and may or may not retain the capacity to proliferatefurther.

As used herein, the term “without the formation of a pluripotentintermediate cell” refers to the transdifferentiation of one cell typeto another cell type, preferably, in one step; thus a method thatmodifies the differentiated phenotype or developmental potential of acell without the formation of a pluripotent intermediate cell does notrequire that the cell be first dedifferentiated (or reprogrammed) andthen differentiated to another cell type. Instead, the cell type ismerely “switched” from one cell type to another without going through aless differentiated phenotype. Accordingly, transdifferentiation refersto a change in the developmental potential of a cell whereby the cell isinduced to become a different cell having a similar developmentalpotential, e.g., a liver cell to a pancreatic cell, a pancreatic alphacell into a pancreatic beta cell, etc. The system and methods of theinvention are well suited for transdifferentiation of cells.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, translation, folding, modification and processing.“Expression products” include RNA transcribed from a gene, andpolypeptides obtained by translation of mRNA transcribed from a gene. Insome embodiments, an expression product is transcribed from a sequencethat does not encode a polypeptide, such as a microRNA.

As used herein, the term “transcription factor” refers to a protein thatbinds to specific parts of DNA using DNA binding domains and is part ofthe system that controls the transcription of genetic information fromDNA to RNA.

As used herein, the term “small molecule” refers to a chemical agentwhich can include, but is not limited to, a peptide, a peptidomimetic,an amino acid, an amino acid analog, a polynucleotide, a polynucleotideanalog, an aptamer, a nucleotide, a nucleotide analog, an organic orinorganic compound (e.g., including heterorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

The term “exogenous” as used herein refers to a nucleic acid (e.g., asynthetic, modified RNA encoding a transcription factor), or a protein(e.g., a transcription factor) that has been introduced by a processinvolving the hand of man into a biological system such as a cell ororganism in which it is not normally found, or in which it is found inlower amounts. A factor (e.g., a synthetic, modified RNA encoding atranscription factor, or a protein, e.g., a polypeptide) is consideredexogenous if it is introduced into an immediate precursor cell or aprogeny cell that inherits the substance. In contrast, the term“endogenous” refers to a factor or expression product that is native tothe biological system or cell (e.g., endogenous expression of a gene,such as, e.g., SOX2 refers to production of a SOX2 polypeptide by theendogenous gene in a cell). In some embodiments, the introduction of oneor more exogenous factors to a cell, e.g., a developmental potentialaltering factor, using the compositions and methods comprisingsynthetic, modified RNAs described herein, induces endogenous expressionin the cell or progeny cell(s) thereof of a factor or gene productnecessary for maintenance of the cell or progeny cell(s) thereof in anew developmental potential.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found, or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally, the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell or population of cells from which itdescended) was isolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a “substantially pure”population of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched. In some embodiments, theisolated population is an isolated population of pluripotent cells whichcomprise a substantially pure population of pluripotent cells ascompared to a heterogeneous population of somatic cells from which thepluripotent cells were derived.

As used herein, the terms “synthetic, modified RNA” or “modified RNA”refer to an RNA molecule produced in vitro, which comprise at least onemodified nucleoside as that term is defined herein below. Methods of theinvention do not require modified RNA. The synthetic, modified RNAcomposition does not encompass mRNAs that are isolated from naturalsources such as cells, tissue, organs etc., having those modifications,but rather only synthetic, modified RNAs that are synthesized using invitro techniques. The term “composition,” as applied to the terms“synthetic, modified RNA” or “modified RNA,” encompasses a plurality ofdifferent synthetic, modified RNA molecules (e.g., at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 25, at least 30, at least 40, at least 50, at least 75, atleast 90, at least 100 synthetic, modified RNA molecules or more). Insome embodiments, a synthetic, modified RNA composition can furthercomprise other agents (e.g., an inhibitor of interferon expression oractivity, a transfection reagent, etc.). Such a plurality can includesynthetic, modified RNA of different sequences (e.g., coding fordifferent polypeptides), synthetic, modified RNAs of the same sequencewith differing modifications, or any combination thereof.

As used herein, the term “polypeptide” refers to a polymer of aminoacids comprising at least 2 amino acids (e.g., at least 5, at least 10,at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 125, at least150, at least 175, at least 200, at least 225, at least 250, at least275, at least 300, at least 350, at least 400, at least 450, at least500, at least 600, at least 700, at least 800, at least 900, at least1000, at least 2000, at least 3000, at least 4000, at least 5000, atleast 6000, at least 7000, at least 8000, at least 9000, at least 10,000amino acids or more). The terms “protein” and “polypeptide” are usedinterchangeably herein. As used herein, the term “peptide” refers to arelatively short polypeptide, typically between about 2 and 60 aminoacids in length.

Microarrays and particularly “cell arrays” or “cell panels” arecurrently needed for screening of large biomolecule libraries, such asRNAs, DNAs, proteins and small molecules with respect to theirbiological functions and for fundamental investigation of cell andgene-functions. Many research facilities both in academia and inindustry need advanced high-density arrays to improve theirscreening-efficiency, velocity and quality. Many screens will firstbecome possible or significantly more affordable with the development ofnext generation microarrays and cell arrays/cell panels, respectively.An invention array or cell panel should typically fit onto a customarymicrotiter scaled plate to ensure the usability of conventionalmicroplate handling robots and microscopes. In one embodiment cellarrays or cell panels can comprise any collection of cell lines thatneed to be assayed as a unit under identical conditions, for examplewhere the only variable is the genotype of the cell lines. An examplecould be a collection of normal and disease specific iPSC lines, ortheir differentiated derivatives, plated in microtiter plates in wellsadjacent to each other. The cell panels may comprise cells derived frommultiple individuals in any “population of interest” and can be selectedsuch that the cells (or individuals from whom the cells are derived) arerepresentative of the diversity of that population of interest. Thecells in the cell panels may be stem cells, differentiated cells madefrom such stem cells, or differentiated cells made bytrans-differentiation from cells of another type (e.g., bytrans-differentiation). Such cell panels can be used, for example, toprobe the activity of a single factor (e.g., small molecule) on multiplegenotypes simultaneously to discover genotype specific effects of thatfactor using the appropriate assays.

One advantage of the present invention is that it provides methods andsystems for generating an essentially limitless supply of isogenic orsynegenic human cells (such as iPSCs and differentiated cells derivedtherefrom) that may be suitable for transplantation, use in drugdiscovery assays, and/or for disease modeling. Such cells (such asiPSCs) may be tailored specifically to the patient, therefore,potentially obviating the significant problem associated with currenttransplantation methods, such as, rejection of the transplanted tissue,which may occur because of host versus graft or graft versus hostrejection. When utilized for drug discovery the cells demonstrate eachperson's response to chemicals when used in drug discovery or theirindividual manifestation of diseases in disease models. Several kinds ofiPSCs or fully differentiated somatic cells prepared from iPSCs derivedfrom somatic cells derived from humans can be stored in an iPSC bank asa library of cells, and one kind or more kinds of the iPSCs in thelibrary can be used for preparation of somatic cells, tissues, or organsthat are free of rejection by a patient to be subjected to stem celltherapy.

In one embodiment the present invention provides methods of makingpanels of cells or “cell panels” that are derived from multipleindividuals in a “population of interest” and that may, in someembodiments, be representative of the diversity of that population ofinterest. Such cell panels can be made, for example, using the automatedsystems of the present invention or other suitable systems known theart. The cells in the cell panels may be somatic cells, stem cells,differentiated cells made from such stem cells, or differentiated cellsmade by trans-differentiation from cells of another type (e.g., bytrans-differentiation). Such cell panels can be made in multiple formatsand can be frozen and stored to form frozen banks of cell panels. Suchcell panels may be useful for a variety of different applications,including, but not limited to, use in assays designed to screen for newdrugs that might be effective in a given population of interest, and/orto test the efficacy, safety, and/or toxicity of drugs in a givenpopulation of interest.

The cell panels of the present invention may include samples obtainedfrom, and/or be designed to be representative of, any “population ofinterest” desired, including, but not limited to, the world population,the population of a particular country, the population of a particularcontinent, the population of a particular geographic region (e.g.,Northern Italian, Indian sub-continent, etc.), the population of aparticular racial or ethnic group (e.g., Ashkenazi Jews, Maoris, etc.),a population of a particular age, a population of a particular sex (maleor female), a population having a particular disease or disorder (e.g.,a specific cancer, metastatic cancer, Huntington disease, Parkinson'sdisease, psoriasis, asthma, post-traumatic stress disorder, traumaticbrain injury, autism, or any other disease or disorder of interest), apopulation having a particular mutation, a population having aparticular genotype, a population having a particular phenotype, apopulation having a particular HLA type, a population having aparticular blood group, a population having a particular drug responseprofile, and the like. In some embodiments the panels of the presentinvention may be designed to be representative of the population ofinterest (e.g., in terms or race, ethnicity, sex, age, genotype,phenotype or any other desired characteristic), for example based onpopulation Census data. In some embodiments the cell panels may compriseengineered lines, such as those created to test for the effects ofparticular mutations.

For some applications it will be desirable to use “control” panels ofcells, or panels comprising “control” cells. Such controls can be usedfor comparison to cells from the populations of interest. For example,if a panel comprises cell samples having a particular mutation (such asa mutation related to a particular disease) it may desirable to have acontrol panel comprising control cell samples, or to include controlcell samples in the panel. In some embodiments, such control cellssamples may comprise isogenic control cell samples, such as cell samplesin which a mutation has been corrected.

In some embodiments the present invention provides panels of stem cellsthat are derived from multiple individuals in a population of interest.In some embodiments the stem cells may comprise induced pluripotent stemcells (iPSCs). Such panels can be made, for example, by obtainingdifferentiated somatic cells from an adult or child and using iPSCmethods known in the art to convert those cells to pluripotent stemcells, for example using the automated systems of the invention. In someembodiments the stem cells may comprise embryonic stem cell (ESCs), forexample ESCs derived from donated embryos (such as those created in anIVF procedure) or ESCs made by a nuclear transfer technique.

In some embodiments the present invention provides panels ofdifferentiated cells that are derived from multiple individuals in apopulation of interest. Types of differentiated cells that may beprovided using in the format of a panel according to the inventioninclude, but are not limited to: oligodendrocytes, beta cells, corticalneurons, dopaminergic neurons, cardiomyocytes, and cells of certainmesenchymal lineages (osteoblasts).

In some embodiments the cell panels of the present invention are madein, and/or provided in, the form of tissue culture vessels, such astissue culture plates, bottles, or vials. In one embodiment the cellpanels of the present invention are made in, and/or provided in,microtiter plates, such as those having 6, 24, 96, 384, 1536, 3456 or9600 wells in one plate. In some embodiments, each well in such amicrotiter plate may comprise cells derived from one individual (such asone human individual) with every well containing cells from differentindividuals. For example, 96 different individuals may be represented inone 96-well plate, 384 different individuals may be represented in one384-well plate, and 1596 different individuals may be represented in one1596-well plate within the population group of interest. In otherembodiments multiple wells within one plate may comprise cells from thesame individual.

In one embodiment the panels of the present invention are made using anautomation platform, such as that described herein or in U.S. patentapplication Ser. No. 13/691,257, the contents of which are herebyincorporated by reference. In other embodiments these cell panels may bemade manually or by any other suitable means known in the art.

The cell panels of the present invention have a variety of uses. In someembodiments the cell panels can be used for disease modeling, drugscreening, toxicology testing (e.g., for testing the toxicity of drugson specific populations of interest), efficacy studies (e.g., fortesting efficacy of drugs on specific populations of interest), studyingbasic biology, studying developmental biology, for generating cellproducts (e.g., materials generated using cells as “factories”), oridentifying groups of individuals similarly affected by drugs. As such,the cell panels can be used in methods that resemble clinical trials butthat are performed in vitro, allowing drugs to be tested on cells fromlarge cohorts of different individuals. In this way it may be possible,for example, to identify subgroups of individuals that respond in aparticular way to drugs before the drugs are used in clinical trials orare approved and used in the population at large.

The cell panels of the present invention can be provided in variousforms. In one embodiment they can be provided as growing/living cells,for example in the form of a microtiter plate, or plates, of livingcells. Such plates of living cells can be passaged as needed tomaintain, continue or expand the cell panel(s). In some embodiments theplates of cells can be passaged using an automated system such as thatdescribed herein. In some embodiments the cell panels of the presentinvention are provided as frozen cells, for example in one or moreplates or vials. In some embodiments the panels of the present inventionmay be frozen and/or thawed using an automated system such as thatdescribed herein.

In one aspect the present invention provides automated systems suitablefor generating, maintaining and handling a variety types, such as iPSCsand differentiated cells produced therefrom. The invention systemgreatly improves the efficiency and reproducibility of making andhandling standardized iPSC lines and other cell lines. Typically,researchers generate iPSCs by hand, which limits the cells utility dueto researcher variability and an inability to generate large numbers ofcells. The invention system circumvents these problems with a completelyautomated system from receipt of the tissue or cell sample to banking oflarge stocks of well-defined iPSC lines and/or differentiated cellsproduced therefrom. The system allows for consistency in the generationof large numbers of cells from many donors, which will facilitate theuse of iPSC technology to discover treatments and cures for manydiseases. Various embodiments and components of the automated systems ofthe invention are described herein. In addition, each of suchembodiments can be modified by inclusion of a data-driven batchingsystem, or a component thereof, as described in other sections of thispatent application.

In one embodiment, the workflow system of the invention includes anautomated system for generating and isolating iPSCs, comprising: asomatic cell, e.g., fibroblast, plating unit for placing cells on aplate; and an induction unit for automated reprogramming of cells bycontacting the cells on the plating unit with reprogramming factors toproduce iPSCs. In some embodiments the system further comprises adata-driven batching system, or a component thereof. In a furtherembodiment, the invention system includes a sorting unit for selectivelysorting and isolating the iPSCs produced by the induction unit byidentifying iPSC specific markers, including, e.g., surface markers orgreen fluorescent proteins inserted by a transfection vector. Somaticcells can be obtained from cell lines, biopsy or other tissue samples,including blood, and the like.

In another embodiment, the invention provides an automated system forgenerating and isolating differentiated adult cells from stem cells,e.g., iPSCs, embryonic stem (ES) cells or mesenchymal stem (MS) cells,comprising: a stem cell plating unit for placing cells, e.g., iPSCs, ESor MS cells, on a plate; and an induction unit for automatedreprogramming of cells by contacting the cells on the stem cell platingunit with reprogramming factors to produce differentiated adult cells.In some embodiments the system further comprises a data-driven batchingsystem, or a component thereof. In one embodiment, the system furtherincludes a sorting unit for selectively sorting and isolating thedifferentiated adult cells produced by the induction unit by identifyingmarkers specific to the differentiated adult cells.

In yet another embodiment, the invention provides an automated systemfor generating and isolating differentiated adult cells from inducedpluripotent stem cells (iPSCs), comprising: an iPSC plating unit forplacing iPSCs on a plate; and an induction unit for automatedreprogramming of iPSCs by contacting the iPSCs on the iPSC plating unitwith reprogramming factors to produce differentiated adult cells. Insome embodiments the system further comprises a data-driven batchingsystem, or a component thereof. In one embodiment, the system furtherincludes a sorting unit for selectively sorting and isolating thedifferentiated adult cells produced by the induction unit by identifyingmarkers specific to the differentiated adult cells.

The invention provides an automated workflow system for producing iPSCsfrom differentiated adult cells. Broadly, the inventive workflow systemprovides a new workflow system that starts with adult differentiatedcells (e.g., isolated or tissue samples) and results in either iPSCs oradult cells derived from pluripotent cells. In some embodiments theworkflow system comprises a data-driven batching system, or a componentthereof. In one embodiment, the adult differentiated cells arepreferably fibroblasts obtained, e.g., from skin biopsies. The adultfibroblasts are converted into induced pluripotent stem cells (iPSCs) bythe inventive workflow that incorporates automation and robotics. Theinventive workflow system is capable of generating thousands of iPSCs inparallel resulting in an accelerated timeframe, in a period of monthsinstead of the years, which would have previously been required. Theinventive workflow system can be adapted to any cell isolation systemfor starting material and be applied to direct or indirect reprogrammingand transdifferentiation, for example. The inventive workflow systemwill allow production employing cellular arrays of cells from 6, 24, 96,384, 1536 sized arrays, or greater (such as 3456 or 9600 sized arrays).The inventive workflow system is flexible and will allow for multipleiterations and flexibility in cell type and tissue. The descriptionherein is shown with fibroblasts as an illustrative somatic cell. Asnoted herein, other cell types are used in the system. The example isnot meant to be limited in this way.

The Workflow System

The workflow system is broken down into four independently-operatedunits:

(1) Quarantine Somatic Cell Isolation and Growth (System 1);

(2) Quarantine Assay (System 2);

(3) Thawing, Infection and Identification (Systems 3, 4, and 5); and

(4) Maintenance, QC, Expansion, and Freezing. (Systems 6, 7, and 8)

Additionally, an automated −80 storage and retrieval system for storingfibroblasts and final clones in 1.4 mL Matrix screw cap tubes, is partof the system. The systems, and the steps and operations that each unitwill perform, will be described below.

System 1, Part A: Quarantine Somatic Cell Isolation and Growth Workflow,Biopsy Processing Pre-Mycoplasma Test

-   -   1. Technician will plate 40 biopsies per week in 6-well dishes;    -   2. 6-well plates will be maintained in quarantine incubator with        200-plate capacity;    -   3. Periodic confluency checks are performed on an integrated        Cyntellect Celigo Cytometer.

The system components that may be used to perform these automated stepsinclude by way of example, STARlet Manual Load, a Modular Arm for 4/8/12ch./MPH, 8 channels with 1000 μl Pipetting Channels and an iSWAP PlateHandler, all available from Hamilton Science Robotics. If centerfugingis needed or desired, an Agilent VSpin Microplate Centerfuge can beused. The software may be Celigo API Software. The incubator may be aCytomat Incubator. For plate handling a Cytomat 24 Barcode Reader,Cytomat 23 mm Stackers, and a Cytomat 400 mm transfer station may beused. For plate tilting, one may use a MultiFlex Tilt Module. The systemcontroller may be a Dell PG with a Windows XP operating system. Thecarrier package may be a Q Growth Carrier Package.

System 1, Part B: Quarantine Growth Workflow, Mycoplasma Test

-   -   1. Retrieve from incubator to deck of Quarantine Growth STARlet,        remove media from wells to plate for ELISA based mycoplasma        test.    -   2. Manually transfer 96-well assay plates to Quarantine Assay        STARlet.

System 1, Part C: Quarantine Growth Workflow, After Passing MycoplasmaTesting

-   -   1. Expanded fibroblasts distributed into multiple cryovials,        capped, transferred to SAM −80° C.

The system components that may be used to perform these automated stepsmay be selected from the same components used in the Quarantine GrowthWorkflow, except a STARlet Auto Load may be used. A Spectramax L Readermay be used as a spectral acquisition device.

System 2: Quarantine Assay Workflow

1. Test using glow luminescence method, Lonza MycoAlert.

2. Perform luminescence plate read on spectral acquisition device.

The system components that may be used to perform these automated stepsinclude STARlet Manual Load, a Modular Arm for 4/8/12 ch./MPH, 8channels with 1000 μl Pipetting Channels and an iSWAP Plate Handler, allavailable from Hamilton Science Robotics. For luminescence assays theBioTek Synergy HT Reader may be used. The system controller may be aDell PG with a Windows XP operating system. The carrier package may be aQ Growth Carrier Package.

Systems 3, 4, and 5: Thawing, Infection and Identification

Thawing Module & Infection Module

-   -   1. Retrieve cryotubes from SAM −80° C. (61, 190)    -   2. Thaw on warming block (122)    -   3. Decap (Hamilton Capper Decapper) (126)    -   4. Add media to dilute cryoprotectants (122)    -   5. Spin (128)    -   6. Resuspend in plating data (122)    -   7. Plate one sample per well of 6-well (62, 122)    -   8. Move to incubator (130, 132)    -   9. Fibroblasts recover for about 3-4 days    -   10. Confluence check on Cyntellect Celigo Cytometer (124)    -   11. Fibroblast passaging of all wells on the same day for        reprogramming (122)    -   12. In batches, tryspin passage wells (122)    -   13. Count cells on Cyntellect Celigo Cytometer (124)    -   14. Plate a defined number per well on one-to-three wells of a        24-well plate consolidating samples onto as few as 24-well        plates as possible (64, 122)    -   15. Return plates to the incubator overnight (130, 132)    -   16. Retrieve plates and thaw virus in tube format and add to        each well of the fibroblasts in the 24-well plates (130, 122)    -   17. Daily partial media exchanges (122)

Magnetic Sorting Module

-   -   18. Harvest cultures with accutase to single-cell suspension        (134)    -   19. Dilute in staining buffer (134)    -   20. Stain with magnetic beads against fibroblast surface marker        (134)    -   21. Wash step (134)    -   22. Apply to magnet (for Dynal beads) or column (for Miltenyi        system) (134, 136)    -   23. Retrieve non-magnetic fraction to new wells (134)    -   24. Count cells on Cyntellect Celigo Cytometer (124)    -   25. Dilute to appropriate cell density for delivering 1-10 cells        per well to 96-well plate in passaging media (66, 134)    -   26. Retrieve new Matrigel or matrix-coated 96-well plate from        4° C. incubator (142)    -   27. Distribute cells to 96-well matrix plates, number based on        cell count for example, two per plates per infection (66, 134)    -   28. Return plates to incubator (132)    -   29. Daily partial media exchanges (122)

Colony Identification Module

-   -   30. Retrieve 96-well plates from incubator to Colony        identification liquid handler (66, 132, 138)    -   31. Perform live cell stain with pluripotency surface marker        (138)    -   32. Image on Cyntellect Celigo Cytometer (140)    -   33. Identify wells with a single-marker positive colony that has        a sharp colony border (140)    -   34. Techs review hits and select 6 per original sample for        passage and retrieve plate and positive well IDs.    -   35. Cherry-pick wells with single positive colonies (138)    -   36. Retrieve new Matrigel or matrix coated 96-well plate from        4° C. incubator (68, 142)    -   37. Harvest selected wells and passage to new 96-well matrix        plate consolidating clones onto as few plates as possible and        plating each in passaging media (68, 138)    -   38. Daily partial media exchanges (122)

The system components that may be used to perform these automated stepsmay be selected from the same components used in the Quarantine GrowthWorkflow with the addition of one or more CORE 96 PROBEHEAD II 1000 μlmodel probe heads.

Systems 6, 7, and 8: Maintenance, QC, Expansion, and Freezing

Maintenance Module

-   -   39. Will serially-passage clones 1:1 into new 96-well        matrix-coated plates until colony density is high enough (68-72,        160)    -   40. Daily feeding of all plates with ˜75% media exchange with        96-tip head (160)    -   41. Periodic monitoring of colony density and growth rates on        Cyntellect Celigo Cytometer (166)    -   42. Plate replication to produce plates for QC of clones (74-86,        160)    -   43. Goal is to expand clones onto multiple plates for use in        several QC assays to eliminate poorly-performing clones until        left with two-to-three high-quality clones per original sample    -   44. Will also cherry-pick and re-array clones that pass QC steps        as the poor clones are eliminated to consolidate clones onto as        few plates as possible (80, 86, 160)    -   45. Daily feeding throughout this process (160)

QC Module

-   -   46. Harvest cells (74, 150)    -   47. Count cells (164)    -   48. Plate a defined cell number in V-bottom plates (range of        5000-10000 cells/well) in 2-6 replicates per line (84, 150)    -   49. Return to incubator—(1 g aggregation) (172)    -   50. Media exchange after two days (150)    -   51. Incubate for additional 12 days in incubator (172)    -   52. Partial media exchange every two days (150)    -   53. Transfer to nucleic acid prep station to remove media from        wells leaving embryoid bodies in the well (84, 192)    -   54. Resuspend in RNA lysis buffer and combine and mix replicates        for each sample and make plates available for analysis in        Nanostring nCounter assay (84, 192)

Freezing Module

-   -   55. Begins with a 96-well plate after an expansion passage (88)    -   56. Incubate 6 days in incubator (172)    -   57. Partial media exchange every day (154)    -   58. Remove plate from incubator (88, 162)    -   59. Remove media (needs to be complete) (154)    -   60. Add cool Pre-freeze media (diluted matrigel in growth media)        (154)    -   61. Incubate in incubator for 1 h (172)    -   62. Remove media (needs to be complete) (154)    -   63. Addition of cold freezing media—low volume (154)    -   64. Seal plate (88, 164    -   65. Samples taken off-line to −80° C. storage to freeze (190)    -   66. Store in vapor phase Liquid Nitrogen

Cryovial Storage

-   -   67. Begins with a 96-well plate after an expansion passage (90)    -   68. Incubate 6 days (172)    -   69. Daily partial media exchanges (154)    -   70. Passage wells 1:1 to a 24-well plate (92, 154)    -   71. Incubate 6 days (172)    -   72. Daily partial media exchanges (154)    -   73. Passage wells 1:1 to a 6-well plate (94, 154)    -   74. Incubate 4-6 days (172)    -   75. Daily partial media exchanges (154)    -   76. Remove plate from incubator (162)    -   77. Partial media exchange with pre-freeze media (154)    -   78. Incubate in incubator for 1 h (172)    -   79. Harvest cells for freezing as for normal passage (154)    -   80. Move to matrix tubes, two-to-three tubes per well (96, 154)    -   81. Spin and remove media (168, 154)    -   82. Addition of cold freezing media (154)    -   83. Cap tubes (170)    -   84. Samples taken off-line to −80° C. storage (190)

The system components that may be used to perform these automated stepsmay be selected from the same components used in the Quarantine GrowthWorkflow.

The iPSCs of the present invention may be differentiated into a numberof different cell types to treat a variety of disorders by methods knownin the art. For example, iPSCs may be induced to differentiate intohematopoetic stem cells, muscle cells, cardiac muscle cells, livercells, cartilage cells, epithelial cells, urinary tract cells, neuronalcells, and the like. The differentiated cells may then be transplantedback into the patient's body to prevent or treat a condition or used toadvance medical research or in to develop drug discovery assays. Thus,the methods of the present invention may be used to as a treatment or todevelop a treatment for a subject having a myocardial infarction,congestive heart failure, stroke, ischemia, peripheral vascular disease,alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer'sdisease, diabetes, cancer, arthritis, wound healing, immunodeficiency,aplastic anemia, anemia, Huntington's disease, amyotrophic lateralsclerosis (ALS), lysosomal storage diseases, multiple sclerosis, spinalcord injuries, genetic disorders, and similar diseases, where anincrease or replacement of a particular cell type/tissue or cellularde-differentiation is desirable.

In one embodiment, the inventive system can also be used to obtain cellpopulations enriched in fully reprogrammed cells, from among cells thathave undergone differentiation in established iPSC cell lines that werecultured under both murine embryonic fibroblast (MEF) feeder layer, aswell as feeder reconditions. The inventive system further enables thelive-sorting of defined subpopulations of fully-reprogrammed, ordifferentiated, iPSC cells into 96-well plates for use inhigh-throughput screening campaigns.

FIG. 1 shows the steps performed by System 1, including plating of abiopsy (2), outgrowth and passaging (4) (rolling production on liquidhandling robot), QC (6) (automated testing for mycoplasma), and (8)automated freezing on liquid handling robot.

FIG. 2 shows the steps performed by Systems 2, 3, and 4. Fibroblasts areplated by the automated system (10), reprogramming factors areintroduced by the automated system (12), iPSCs are isolated by automatedsorting and isolation (14), desired clones are selected and expanded bythe automated system (16), automated quality checks (QC) for pluripotentstatus by marker assays and embryoid body assays (18), followed byautomated freezing and storage of desired cells (20).

FIG. 3 is a flowchart showing the step (22) through (60) involved inSystem 1.

FIG. 3 illustrates an example of the workflow and decision tree forproduction of fibroblasts from biopsies. The workflow is divided intoQuarantine (58) and Clean phases (60). As biopsies enter the facility, atechnician plates biopsies in 6-well plates (22) and logs the platesinto the automated incubator (24). After biopsies are given time toattach to the plate, the liquid handling robot retrieves the plates fromthe automated incubator to feed and check confluency of the outgrowthson an automated microscope (26). The plates are returned to theincubator and allowed to outgrow (28). The liquid handler removes theplate from the incubator and exchanges the media for antibiotic andantimycotic free media (30). The robot moves the plate to the incubatorfor another five days (32). The robot then removes the plate andretrieves media to daughter plates for mycoplasma test (34). Thedaughter plates are moved to the Quarantine Assay system for mycoplasmatesting (36). A choice is then made based on a positive signal from theassay (38). If all wells of a 6-well plate fail with a positivemycoplasma assay result (40) they are discarded. If all wells of a6-well plate are negative and free of mycoplasma, they are transferredout of quarantine into the clean growth system (46). If some wells arepositive and some wells are negative, the negative wells are maintainedin quarantine (42). The negative wells are passaged (44) to new plates,transferred to the incubator, and the source plates containing positivewells are discarded. These cultures proceed through steps to retest formycoplasma (24, 26, 28, 30, 32, 34, 36, 38). Clean cultures aremonitored for growth (50), passaged (52) and frozen in cryovials (54,56).

FIGS. 4A, 4B1, 4B2, and 4C illustrate an example of the flow of patientsamples through multi-well tissue culture plates during the automatedreprogramming process. At the top of each diagram, a flowchart describesthe flow of procedures performed at each step of the workflow (70, 88,98). At the bottom of each diagram, multi-well cell culture plates areshown with platemaps for example samples represented by shaded wells orgroups of wells marked with sample labels (61-68, 72-86, 88-96).Transfer of a sample from plate-to-plate or well-to-well through theprocedure is shown from left to right as indicated by arrows. As shownin FIG. 4A, the automated iPSC derivation process begins when patientsamples and control fibroblast samples (61) are plated in individualwells of a 6-well plate (62). These are passaged at defined cell numberinto individual wells of a 24-well plate (64) for infection usingviruses encoding reprogramming factors or other means of introducingreprogramming factors to the cells. In the next step, reprogrammedsamples are depleted of non-reprogrammed cells by cell sorting or, as ispreferred, using magnetic bead based enrichment and plated at clonaldensity in multiple wells in 96-well plates (66). Two such plates areshown in this example. In this example, 6 wells, as indicated by wellswith a dot in the middle (66) are identified containing a single clonepositive for a pluripotency surface marker as assayed byimmunofluorescent analysis on automated imager. These clones arepassaged and cherry picked to reformat the clones into a minimum numberof 96-well plates (68). The example figure shows six clones perindividual starting sample and indicates that clones from 16 startingsample can be arrayed onto a 96-well plate. To facilitate plateprocessing, this cherry picking step can be performed over multiplepassages to consolidate the clones onto a minimum number of plates. Asshow in FIGS. 4B1 and 4B2, these clones are serially passaged untilconfluence of stem cell colonies within a well is achieved for eachstarting sample (72). Each plates' samples are then replicated ontoduplicate plates (74-86), to allow for the quality control (6) andselection of clones that demonstrate appropriate stem cellcharacteristics. To begin the QC process, one plate is generated by thesystem for a Pluripotency quality control assay needed to determinepluripotent status of the individual clones (74) and one plate isgenerated for carrying forward in subsequent passages (76). The platethat is carried forward is passaged again into three plates (78, 80, 82)for further quality control and expansion. One plate is harvested for QCassays to characterize Karyotype and genetic diversity (78). A secondplate (82) is passaged onto v-bottom plates to form embryoid bodies (84)for a QC assay that assesses differentiation capability of the iPSclones. The final plate (80) is carried forward for further expansion.Individual clones that do not pass quality control from previouspluripotency QC assays are not carried forward as shown by the “X” inthe wells indicated in FIGS. 4A, 4B1, 4B2 and 4C. In the example shownin FIG. 4B2, the consolidated plate (86) will contain iPS lines (ordifferentiated lines) from up to 32 individuals represented by 3 iPSclones per individual on a single 96 well plate or up to 96 individualsif represented by a single clone each. Remaining clones are consolidatedonto as few plates as possible until one to three clones remain (86-92).As shown in FIG. 4C, these are expanded for cryopreservation whileattached to the plate (88) or further expanded (92-94) and cryopreservedin cryovials (96). Any or all information from the pluripotency markerscreen shown in FIG. 4A (70), and the quality control assays shown inFIG. 4B1 can be used alone or in combination to decide which clones toselect for consolidation and arraying in the automated process.

Methods for transfecting and transforming or reprogramming adult cellsto form iPSC lines are generally known, e.g., Takahashi et al., 2007Cell, 131: 861-872, 2007, Yu et al., 2007, Science, vol. 318, pp.1917-1920. iPSC are induced from somatic cells with reprogrammingfactors. Reprogramming factors are contemplated to include, e.g.,transcription factors. The method for reprogramming adult cellsincludes, e.g., introducing and expressing a combination of specifictranscription factors, e.g., a combination of Oct3/4, Sox2, Klf4 andc-Myc genes. Others have demonstrated that other transcription factorsmay be employed in transforming or reprogramming adult cells. Theseother transcription factors include, e.g., Lin28, Nanog, hTert and SV40large T antigen as described, for example, by Takahashi et al., 2006Cell, 126: 663-676 and Huiqun Yin, et al. 2009, Front. Agric. China3(2): 199-208, incorporated by reference herein.

In another aspect, iPSCs can be generated using direct introduction ofRNAs into a cell, which, when translated, provide a desired protein orproteins. Higher eukaryotic cells have evolved cellular defenses againstforeign, “non-self,” RNA that ultimately result in the global inhibitionof cellular protein synthesis, resulting in cellular toxicity. Thisresponse involves, in part, the production of Type I or Type IIinterferons, and is generally referred to as the “interferon response”or the “cellular innate immune response.” The cellular defenses normallyrecognize synthetic RNAs as foreign, and induce this cellular innateimmune response. In certain aspects where the ability to achievesustained or repeated expression of an exogenously directed proteinusing RNA is hampered by the induction of this innate immune response,it is desirable to use synthetic RNAs that are modified in a manner thatavoids or reduces the response. Avoidance or reduction of the innateimmune response permit sustained expression from exogenously introducedRNA necessary, for example, to modify the developmental phenotype of acell. In one aspect, sustained expression is achieved by repeatedintroduction of synthetic, modified RNAs into a target cell or itsprogeny. The inventive methods include natural or synthetic RNAs.

The natural, modified, or synthetic RNAs in one aspect, can beintroduced to a cell in order to induce exogenous expression of aprotein of interest in a cell. The ability to direct exogenousexpression of a protein of interest using the modified, synthetic RNAsdescribed herein is useful, for example, in the treatment of disorderscaused by an endogenous genetic defect in a cell or organism thatimpairs or prevents the ability of that cell or organism to produce theprotein of interest. Accordingly, in some embodiments, compositions andmethods comprising the RNAs described herein can be used for thepurposes of gene therapy.

The RNAs described can advantageously be used in the alteration ofcellular fates and/or developmental potential. The ability to express aprotein from an exogenous RNA permits either the alteration or reversalof the developmental potential of a cell, i.e., the reprogramming of thecell, and the directed differentiation of a cell to a moredifferentiated phenotype. A critical aspect in altering thedevelopmental potential of a cell is the requirement for sustained andprolonged expression of one or more developmental potential alteringfactors in the cell or its immediate progeny. Traditionally, suchsustained expression has been achieved by introducing DNA or viralvectors to a cell. These approaches have limited therapeutic utility dueto the potential for insertional mutagenesis.

One of the areas that can most benefit from the ability to express adesired protein or proteins over a sustained period of time fromexogenous RNAs as described herein is the generation of pluripotent ormultipotent cells from cells initially having a more differentiatedphenotype. In this aspect, RNAs encoding a reprogramming factor orfactors are used to reprogram cells to a less differentiated phenotype,i.e., having a greater developmental potential.

A major goal of stem cell technology is to make the stem celldifferentiate into a desired cell type, i.e., directed differentiationor produce cells via transdifferentiation. Not only are the compositionsand methods described herein useful for reprogramming cells, they arealso applicable to this directed differentiation andtransdifferentiation of cells to a desired phenotype. That is, the sametechnology described herein for reprogramming is directly applicable tothe differentiation of the reprogrammed cell, or any other stem cell orprecursor cell, for that matter, to a desired cell type.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule comprises at least twomodified nucleosides. In one such embodiment, the two modifiednucleosides are selected from the group consisting of 5-methylcytidine(5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U),2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine(Um), 2′ deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine(m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2,N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In one such embodiment of this aspect and all suchaspects described herein, the at least two modified nucleosides are5-methylcytidine (5mC) and pseudouridine. (see e.g., Rossi US2012/0046346, herein incorporated by reference).

Genes, proteins or RNA used in the methods of the invention include butare not limited to OCT4, SOX1, SOX 2, SOX 3, SOX15, SOX 18, NANOG, KLF1,KLF 2, KLF 4, KLF 5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2, TERT, and LIN28.

It has also been shown that a single transcription factor may beemployed in reprogramming adult fibroblasts to iPSCs with the additionof certain small molecule pathway inhibitors. Such pathway inhibitorsinclude e.g., the transforming growth factor-beta (TGFb) pathwayinhibitors, SB431542(4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide),and A-83-01[3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide],the extracellular signal-regulated kinases (ERK) andmicrotubule-associated protein kinase (MAPK/ERK) pathway inhibitorPD0325901(N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide),the GSK3 inhibitor CHIR99021[6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile]which activates Wnt signaling by stabilizing beta-catenin, thelysine-specific demethylasel Parnate (a/k/a tranylcypromine), the smallmolecule activator of 3′-phosphoinositide-dependent kinase-1 (PDK1) PS48[(2Z)-5-(4-Chlorophenyl)-3-phenyl-2-pentenoic acid], the histonedeacetylase (HDAC) inhibitors sodium butyrate and valproic acid, smallmolecules that modulate mitochondrial oxidation (e.g.,2,4-dinitrophenol), glycolytic metabolism (fructose 2,6-bisphosphate andoxalate), HIF pathway activation (N-oxaloylglycine and Quercetin) Zhu etal., 2010, Cell Stem Cell 7: 651-655, incorporated by reference hereinit its entirety. Zhu et al showed that Oct4 combined with Parnate andCHIR99021 was sufficient to reprogram adult human epidermalkeratinocytes.

Although individual protocols differ, a general reprogramming protocolconsists of expanding differentiated adult cells from tissue samples,e.g., skin biopsies and contacting them with reprogramming factors asdiscussed above, e.g., infecting them, i.e., transfecting, with e.g.,expression vectors, such as viral constructs containing transcripts forpluripotent transcription factors. The fibroblasts are obtained byart-known methods, e.g., by mechanically disrupting the tissue followedby enzymatic dissociation to release the fibroblasts, and culturing thefibroblasts by art-known methods, e.g., as described by Dimos et. al.,2008, Science Vol. 321 (5893): 1218-1221.

While illustrative aspects of the invention use vectors, e.g., viralvectors, plasmid vectors, in some aspects vectors are not required fortransfection techniques, including those transferring mRNA molecules tocells.

Transfection of the fibroblasts with an expression vector is carried outaccording to instructions provided with the desired vector. After a time(e.g., ranging from about 2 to about 10 days post-transfection, thecells are dissociated and contacted with fluorescent tagged antibodiesraised against the CD13^(NEG), SSEA4^(POS) and Tra-1-60^(POS) surfacemarkers. The dissociated and antibody-labeled cells are then resuspendedin a phosphate buffered saline solution and moved to an automatedsorting and isolation of iPSC clones. Surface marker positive cells aresorted by tag color or absence thereof directly into sterile tubescontaining tissue culture media or multi-well (6-96 well) tissue cultureplates coated with MEFs or cell free biological matrices and cultureduntil formation of visible colonies occurs.

Colonies are then further confirmed as iPSC by light microscopicinspection of the resulting clones or optionally by microscopicfluorescence inspection of clones labeled with fluorescent taggedantibodies. Optionally, in certain embodiments, one or more of thevectors also insert a green fluorescence protein (GFP) expressionmarker, for convenience in sorting and identification. Severalindividual colonies possessing morphological characteristics consistentwith pluripotent ES cell lines are plucked from cultures and expandedindividually to form monoclonal cultures.

In some embodiments of the present invention cells are subjected toanalysis to provide early confirmation and identification of iPSCs.Preferably, such analysis is conducted by Southern blot, or otherart-known methods which include, but are not limited, to MicroArray,NanoString, quantitative real time PCR (qPCR), whole genome sequencing,immunofluorescence microscopy, flow cytometry, and fluorescenceactivated cell sorting.

In one embodiment detection of enzymatic activity of alkalinephosphatase, positive expression of the cell membrane surface markersSSEA3, SSEA4, Tra-1-60, Tra-1-81 and the expression of the KLF4, Oct3/4,Nanog, Sox2 transcription factors in, for example, presumptivelyreprogrammed human fibroblasts, confirms that a clone is an iPSC. In oneembodiment all of the markers are present, but in some embodiments asubset of the markers are present.

In another embodiment positive expression of the cell membrane surfacemarkers SSEA4 and Tra-1-60 and negative expression of CD13 provides animproved method for identifying reprogrammed human fibroblasts andconfirming that a clone is an iPSC. This improved system is described inmore detail in Example 3, whereby fluorescence activated cell sorting(FACS) is used to identify and isolate cells/clones that areCD13-negative, SSEA4-positive and Tra-1-60-positive resulting inimproved yield/selection of reprogrammed IPSCs and depletion of bothparental and contaminating partially reprogrammed cells.

In some aspects the present invention provides “gene sets” comprisinggenes whose expression can be used to detect, or confirm the presence orgeneration of, particular cells such as iPSCs or other pluripotent cellsor differentiated cells derived from such iPSCs or other pluripotentcells. Such gene sets, and methods and compositions (such as probesand/or other detection agents) that allow detection of the expression ofgenes from such gene sets, can be used in a variety of differentsituations. For example they can be used in accordance with theautomated systems described herein (for example as part of a colonyidentification step or a quality control step), or they can be used inany other situation in which it is desired to detect, or confirm thepresence or generation of pluripotent stem cells, such as iPSCs, ordifferentiated cells produced therefrom.

In one embodiment the present invention provides the “Pluri25” gene set,and nucleic acid probes or other agents (such as antibodies) capable ofdetecting expression of genes in the Pluri25 gene set (which may bereferred to as a Pluri25 probe set). Such a gene/probe set may be usedto detect, or confirm the presence or generation of, iPSCs. The Pluri25gene/probe set comprises the following genes, or probes or other agentsfor detection of the expression of the following genes: four retrovialtransgenes (tOct4, tSox2, tKlf4, and tC-Myc), four Sendai transgenes(tOct4, tSox2, tKlf4, tC-Myc) plus Sendai vector marker (SeV), sevenpluripotency markers (POU5F1 (Oct4), SOX2, KLF4, MYC, LIN28, NANOG, andZFP42), three spontaneous differentiation markers (SOX17, AFP, andNR2F2), one fibroblast marker (ANPEP (CD13)), three house-keepingmarkers (ACTB, POLR2A, and ALAS1) and two sex markers (SRY and XIST)—fora total of 25 markers. The genes in the Pluri25 gene set are also listedin Table 1, below:

TABLE 1 Pluri25 Gene Set Sendai Pluripotency Spontaneous Retroviraltransgenes Markers Differentiation Fibroblasts Housekeeping tOct4S-tOct4 POU5F1 SOX17 ANPEP ACTB (OCT4) (CD13) tSox2 S-tKlf4 SOX2 AFPPOLR2A tKlf4 S-tC-myc KLF4 NR2F2 ALAS1 tC-Myc S-tSox2 MYC SeV LIN28NANOG ZFP42

A Pluri25 probe set may comprise nucleic acid probes or other agents(such as antibodies) capable of detecting expression or expressionproducts of each of the genes in the Pluri25 gene set. Individual probesor detection agents may be used for each gene or, where appropriate,single probes or detection agents spanning several of the genes or geneexpression products may be used. For example, a single nucleic acidprobe spanning all of the four retroviral transgenes may be used. Thesequences of each of the genes in the Pluri25 gene set are known in theart, and nucleic acid probes or other detection agents (such asantibodies) capable of detecting expression of each of these genes maybe available in the art or may be made using standard methods known inthe art. The Pluri25 gene set or probe set can be used to monitorpluripotency in human stem cell cultures, analyze contamination withdifferentiated cells or human fibroblasts, monitoring the sex of thecells, and monitor expression of retroviral and/or Sendai transgenes orvector components. The Pluri25 gene or probe set also contains a probe(SeV) for monitoring Sendai virus expression independent of expressionof any transgenes. Further description of the Pluri25 gene/probe set,including validation studies and other data generated using the Pluri25gene/probe set, is provided in Example 3, and Table 1.

In some embodiments variations on the Pluri25 gene or probe set may beused. For example, in some embodiments the Pluri25 gene or probe set maybe modified so as to exclude the retroviral transgene markers but keepthe Sendai transgene and/or Sendai vector markers. In other embodiments,the Pluri25 gene or probe set may be modified so as to exclude theSendai transgene and/or Sendai vector markers but keep the retrovialtransgene markers. In other embodiments the Pluri25 gene or probe setmay be modified so as to exclude both the Sendai transgene/vectormarkers and the retrovial transgene markers. Thus, in one embodiment,the present invention provides a gene or probe set comprising: sevenpluripotency markers (POU5F1 (Oct4), SOX2, KLF4, MYC, LIN28, NANOG, andZFP42), three spontaneous differentiation markers (SOX17, AFP, andNR2F2), one fibroblast marker (ANPEP (CD13)), three house-keepingmarkers (ACTB, POLR2A, and ALAS1) and two sex markers (SRY and XIST)—for a total of 16 markers.

In another embodiment the “3GLSC100” gene set, and in particular nucleicacid probes or other agents (such as antibodies) capable of detectingexpression products of genes in the 3GLSC100 gene set (which may bereferred to as a 3GLSC100 probe set) may be used to detect, or confirmthe presence or generation of iPSCs (similarly to the Pluri25 gene set)and can also be used to monitor differentiation of pluripotent stemcells by embryoid body assays (either directed or undirected) and can beused in accordance with the analysis methods described by Bock et al.(2011) “Reference Maps of human ES and iPS cell variation enablehigh-throughput characterization of pluripotent cell lines,” Cell 144:439-452, the contents of which are hereby incorporated by reference. The3GLSC100 gene set comprises 83 genes selected from among the publishedgermlayer scorecard of Bock et al. and 17 additional genes that are asubset of the Pluri25 gene set. The genes in the 3GLSC100 gene set arelisted in Table 2 below.

TABLE 2 3GLSC100Gene Set Mesoderm Ectoderm Endoderm Retroviral SendaiPluripotent Other Housekeeping ABCG2 ABCG2 APOE tOct4 tOct4 POU5F1 SRYACTB ADIPOQ APOE CD44 tSox2 tSox2 NANOG XIST POLR2A ANPEP CD44 CDH2tKlf4 tKlf4 ZFP42 ALAS1 CD34 CDH2 CDX2 tC-Myc tC-Myc CD36 CRABP2 CTNNB1SeV CD4 EN1 FOXA2 CD44 FAS GATA4 CDH1 FGFR2 GATA6 CDH2 FUT4 GCG CDH5GATA2 HNF1A CEACAM1 GATA3 HNF1B DLL1 HAND1 ISL1 FUT4 ICAM1 ITGA6 GATA3ITGA4 ITGB1 GATA4 ITGA6 NEUROG3 HHEX ITGB1 NKX2-5 ICAM1 MAP2 PAX6 INHBAMAPT PDX1 ITGA4 MCAM SLC2A2 ITGA6 MNX1 SST ITGAL NCAM1 SYP ITGAM NEFLTHY1 ITGAV NES ITGAX NEUROG3 ITGB1 NGFR ITGB3 NOG KDR NOTCH1 KIT OTX2LEF1 PAX3 MCAM PAX6 MME PAX7 MYOD1 PDGFRA MYOG SNAI2 NCAM1 SOX10 NESSOX2 NGFR SOX9 NOTCH1 SYP PECAM1 TDGF1 SDC1 TH SPI1 THY1 SRF STAT3 TTHY1 TNFRSF1A TWIST1

A 3GLSC100 probe set may comprise nucleic acid probes or other agents(such as antibodies) capable of detecting expression or expressionproducts of each of the genes in the 3GLSC100 gene set. Individualprobes or detection agents may be used for each gene or, whereappropriate, single probes or detection agents spanning several of thegenes or gene expression products may be used. For example, a singlenucleic acid probe spanning all of the four retroviral transgenes in the3GLSC100 gene set may be used. The sequences of each of the genes in the3GLSC100 gene set are known in the art, and nucleic acid probes or otherdetection agents (such as antibodies) capable of detecting expression ofeach of these genes may be available in the art or may be made usingstandard methods known in the art. The 3GLSC100 gene set or probe setcan be used in the same ways that the Pluri25 gene set is used and canalso be used (as described above). Further description of the 3GLSC100gene/probe set, including validation studies and other data generatedusing the 3GLSC100 gene/probe set, is provided in Example 3.

In some embodiments variations on the 3GLSC100 gene or probe set may beused. For example, in some embodiments the 3GLSC100 gene or probe setmay be modified so as to exclude the retroviral transgene markers butkeep the Sendai transgene and/or Sendai vector markers. In otherembodiments, the 3GLSC100 gene or probe set may be modified so as toexclude the Sendai transgene and/or Sendai vector markers but keep theretrovial transgene markers. In other embodiments the 3GLSC100 gene orprobe set may be modified so as to exclude both the Sendaitransgene/vector markers and the retrovial transgene markers.

In another embodiment the “cardiac 1” or “cardiac 2” gene set(collectively the “cardiac gene sets”), and in particular nucleic acidprobes or other agents (such as antibodies) capable of detectingexpression products of genes in such gene sets (which may be referred toas a cardiac probe sets) may be used to detect, or confirm the presenceor generation of, cells that are on a path towards differentiation intocardiomyocytes, such as cells that are have been derived from iPSCs orother pluiropotent cells and have been treated to encouragedifferentiation down a cardiomyocyte lineage. The “cardiac 1” gene setcomprises the following genes: ACTN1, BMP4, GATA4, GJA1, IRX-4, ISL1,KDR, MEF2A, MEF2C, MESP1, MYH6, MYH7, MYL2, MYL7, NKX2-5, NPPA, PDGFRa,SIRPA, TBX20, TBX5, TNNI3, TNNT2, VCAM1, VWF, MIXL1, NANOG, OCT4, SOX17,Brachury T and KCNJ2—for a total of 30 genes. The “cardiac 2” gene setcomprises the all of the genes in the cardiac 1 gene set and thefollowing four additional genes: GAPDH, GUSB, HPRT1, and TBP—for a totalof 34 genes.

A cardiac probe set may comprise nucleic acid probes or other agents(such as antibodies) capable of detecting expression or expressionproducts of each of the genes in the cardiac gene set. Individual probesor detection agents may be used for each gene or, where appropriate,single probes or detection agents spanning several of the genes or geneexpression products may be used. The sequences of each of the genes inthe cardiac gene set are known in the art, and nucleic acid probes orother detection agents (such as antibodies) capable of detectingexpression of each of these genes may be available in the art or may bemade using standard methods known in the art.

The cardiac gene sets or probe sets can be used to establish thedifferentiation stage of pluripotent stem cells when pushed todifferentiate towards a cardiomyocyte phenotype. The cardiac gene setscomprise pluripotency markers, cardiac mesoderm markers, cardiacprogenitor markers, immature cardiomyocyte markers, and maturecardiomyocyte markers. They also include vascular markers and surfacemarkers expressed by cardiomyocytes during differentiation to facilitatepurification, for example by via flow cytometry or by a method usingmagnetic beads. In some embodiments variations on the cardiac 1 andcardiac 2 gene or probe sets may be used.

Any art-known transfection vector may be employed as a reprogrammingfactor, including, e.g., an RNA such as mRNA, microRNA, siRNA, antisenseRNA and combinations thereof. Other expression vectors that may beemployed include, e.g., a retrovirus, a lentivirus, an adenovirus, anadeno associated virus, a herpes virus, a Sindbis virus, a pox virus, abacula virus, a bacterial phage, a Sendai virus and combinationsthereof. Preferably, an employed vector is a non-replicative vector suchas, e.g., Sendai virus vectors engineered to be nonreplicative. Thepreferred Sendai virus vector, while incapable of replication, remainscapable of productive expression of nucleic acids encoding protein(s)carried by the vector, thereby preventing any potential uncontrolledspread to other cells or within the body of a vaccinee. This type ofSendai vector is commercially available as a CytoTune™-iPSC Sendai viralvector kit (DNAVEC, DV-0301).

Any art-known transfection method may be employed to insert such vectorsinto the adult fibroblasts, including, e.g., electroporation, gene gun,and the like. Chemical transfection is optionally conducted by means ofa transfecting agent e.g., a polymer, calcium phosphate, a cationiclipid, e.g., for lipofection, and the like. Cell penetrating peptidesare also optionally employed to carry vectors or other agents into theadult fibroblast cells. In brief, cell-penetrating peptides includethose derived from proteins, e.g., protein transduction domains and/oramphipathic peptides that can carry vectors or other agents into thecell include peptides. The subject of cell-penetrating peptides has beenreviewed, e.g., by Heitz et al., 2009 British Journal of Pharmacology,157: 195-206, incorporated by reference herein in its entirety. Othercell penetrating peptides are art-known, and are disclosed by Heitz, Id.Other cell-penetrating technologies including, e.g., liposomes andnanoparticles, are also contemplated to be employed in the methods ofthe present invention. Liposomes and nanoparticles are also described byHeitz, Id.

Antibodies can be employed in order to identify the transformed cells.Four antibodies against stem cell specific surface proteins are commonlyused to identify and characterize human pluripotent stem cellpopulations; SSEA3, SSEA4, Tra-1-60 and Tra-1-81. The Stage SpecificEmbryonic Antigens 3 and 4 (SSEA3 and SSEA4) are two monoclonalantibodies which recognize sequential regions of a ganglioside presenton human 2102Ep cells (Henderson et al., 2002 Stem Cells 20: 329-337;Kannagi et al., 1983, Embo J2: 2355-2361). The Tra-1-60 and Tra-1-81antibodies were originally raised against human embryonal carcinoma (EC)cells (P W et al., 1984, Hybridoma 3: 347-361) and have been shown tospecifically recognize a carbohydrate epitope on a keratan sulfatedglycoprotein identified as podocalyxin, a member of the CD34-relatedfamily of sialomucins (Badcock et al., 1999, Cancer Research 59:4715-4719; Nielsen et al., 2007, PLoS ONE 2: e237; Schopperle andDeWolf, 2007, Stem Cells 25: 723-730). Several other surface markershave been shown to be expressed on ES cells and include CD326 or EpCam(Sundberg et al., 2009, Stem Cell Res 2: 113-124), CD24 (Heat StableAntigen) and CD133 (Barraud et al., 2007, Journal of NeuroscienceResearch 85, 250-259) (Gang et al., 2007, Blood 109: 1743-1751). Chan etal., 2009, Id. reported that the identification of bona fide IPSc fromfibroblasts undergoing reprogramming via four factor retro viraltransduction can be achieved via live cell imaging and by theobservation, over time, that fibroblasts lose expression of the cellsurface markers CD13 and D7Fib, and gain expression of the pluripotentstem cell markers SSEA4 and Tra-1-60 (Chan et al., 2009, Id.).

Also contemplated to be within the scope of the invention arecompositions comprising iPSCs, e.g., compositions employed as researchtools, or as pharmaceutical compositions, comprising effective amountsof iPSCs prepared by the inventive automated system.

The invention further relates to treating a disease or disorder in ananimal or person in need thereof by administering the iPSCs, e.g.,methods of treatment and/or tissue/organ repair by administering iPSCsproduced by the inventive automated system, or differentiated cellsderived therefrom. Appropriate differentiated cells (of ectodermal,mesodermal or endodermal lineage) may be derived from iPSCs produced bythe inventive methods. The mode of administration can be determined by aperson of skill in the art depending on the type of organ/injury to betreated. For example, iPSCs or differentiated cells derived therefrom,may be administered by injection (as a suspension) or implanted on abiodegradable matrix.

In addition, the invention relates to methods of testing pharmaceuticalsby contacting iPSCs, transdifferentiated, or differentiated cellsderived therefrom, for example, with one or more pharmaceutical agentsof interest, and then detecting the effect of the applied pharmaceuticalagent(s) on the contacted cells. For efficiency, pharmaceutical agent(s)are applied to a battery of iPSCs, or differentiated cells derivedtherefrom. The cells can vary in tissue source, in differentiated celltype, or allelic source, to allow identification of cells or tissuetypes that react favorably or unfavorably to one or more pharmaceuticalagents of interest.

Further, the iPSCs produced by the inventive automated system may beused as a vehicle for introducing genes to correct genetic defects, suchas osteogenesis imperfecta, diabetes mellitus, neurodegenerativediseases such as, for instance, Alzheimer's disease, Parkinson'sdisease, the various motor neuron diseases (MND), e.g., amyotrophiclateral sclerosis (ALS), primary lateral sclerosis (PLS), progressivemuscular atrophy (PMA) and the like.

iPSCs produced by the inventive automated system may also be employed toprovide specific cell types for biomedical research, as well asdirectly, or as precursors, to produce specific cell types forcell-based assays, e.g., for cell toxicity studies (to determine theeffect of test compounds on cell toxicity), to determine teratogenic orcarcinogenic effects of test compounds by treating the cells with thecompound and observing and/or recording the compound's effects on thecells, e.g., effect on cellular differentiation.

The present invention may be better understood by reference to thefollowing non-limiting Examples. The following examples are presented inorder to more fully illustrate the preferred embodiments of theinvention. They should in no way be construed, however, as limiting thebroad scope of the invention.

Example 1 Illustrative Automated Systems

FIGS. 5A, 5B, 5C illustrate an example of the equipment configurationneeded to accomplish the workflow. FIG. 5A shows a system configurationfor the automated expansion and quality control of a fibroblast bank.FIG. 5B shows a system configuration for the automated thawing ofpatient samples, such as fibroblasts, automated introduction ofreprogramming factors with the patient samples, such as fibroblasts,automated cell sorting with MultiMACS, and automated colonyidentification and reformatting. FIG. 5C shows a system configurationfor the automated expansion of iPS clones, automated Embryoid Bodyproduction, and automated freezing.

Automated Derivation of a Fibroblast Cell Bank

As an example, the hardware configuration used to accomplish thederivation of a fibroblast bank consists of a Hamilton STARlet liquidhandling robot (100) connected to the following hardware components: aCytomat 24C GLS automated incubator (108) that allows for the incubationof cell cultures, a Cyntellect Celigo cytometer (102) for automatedimage acquisition and analysis, an Agilent V-Spin automated centrifuge(106) for the centrifugation of cells in plates or tubes, and a HamiltonCapper DeCapper (104) for the automated capping and decapping ofcryotubes. These components are further controlled by programmablesoftware (118) on a PC that communicates with all instruments andcontrols the manipulation of cell culture-ware and cells among thehardware components. The controller software further communicates withscheduling software (120) to link System interactions. The HamiltonSTARlet (100) is equipped with a Modular Arm for 4/8/12 channelpipetting, 8 pipetting channels, iSWAP plate handler, CO-RE Gripper forplate and lid handling, MultiFlex tilt Module for tilting plates duringmedia exchanges, Hamilton Heated Shaker 2.0, as well as a CarrierPackage for flexible layout of the liquid handling platform with plateand lid parks, pipette stackers, daughter plate stackers and troughs forholding media. The Cyntellect Celigo (102) is comprised of an imagingunit and programmable software on a PC for control of image acquisitionand image analysis. The Celigo is preferred because it does not move thecell culture plates during imaging thereby reducing agitation of platedbiopsies. The Hamilton Capper Decapper (104) and the Agilent V-Spincentrifuge (106) are contained with the Hamilton STARlet within a NuAireBSL II biosafety cabinet (110) to maintain a sterile operatingenvironment during manipulation of cell culture plates.

To control plate handling on the automated system, MICROLAB STAR VENUSTWO Base Pack 4.3 software (118) with VENUS Dynamic Scheduler 5.1 (120)are used in conjunction with individual attached hardware componentdrivers for the centrifuge (106), Capper Decapper (104), Celigo (102),and Cytomat 24 (108) and Cytomat transfer station to integrate theoperation of the system. The following methods programmed using theprovided controller software (118) are needed for functionality of thesystem and can be combined in defined sequence to accomplish thederivation of fibroblast lines from patient skin biopsies:

-   -   1. Load 6-well biopsy plates (22, 24) onto the STARlet (100) and        transfer to the Cytomat incubator (108).    -   2. Confluency check (26, 28) on Celigo (102) and a media        exchange on the STARlet (100).

3. Confluency check (28) on Celigo (102).

4. Media change (30) on the STARlet (100) for full media exchange.

5. Assay plate preparation (34) on STARlet (100) and Agilent V-Spincentrifuge (106).

6. Passaging (44) on the STARlet (100).

7. Passage and cherry pick (42) on the STARlet (100).

8. Passage, harvest and freeze on the STARlet (100).

9. Retrieve plates (46, 40) onto the STARlet (100) from the Cytomat(108).

Automated Mycoplasma Testing on Quarantine Assay System

An independent hardware configuration is used to accomplish themycoplasma testing of a fibroblast bank and consists of a HamiltonSTARlet liquid handling robot (112) connected to a BioTek Synergy HTReader (114). These components are further controlled by programmablesoftware (116) on a PC that communicates with all instruments andcontrols the manipulation of cell culture-ware and cells between thehardware components. The Hamilton STARlet (112) is equipped with aModular Arm for 4/8/12 channel pipetting, 8 pipetting channels, iSWAPplate handler, CO-RE Gripper for plate and lid handling, as well as aCarrier Package for flexible layout of the liquid handling platform withplate and lid parks, pipette stackers, daughter plate stackers and plateparks and troughs for holding reagents needed for the assay.

To control plate handling on the automated system, MICROLAB STAR VENUSTWO Base Pack 4.3 software (116) is used in conjunction with theattached hardware component drivers for the BioTek Synergy HT Reader(114) to integrate the operation of the system. A method is programmedusing this software that allows execution of the MycoAlert MycoplasmaDetection assay (36) and data analysis to determine assay result (38).

Automated System for Thawing, Infection, and Identification ofReprogrammed Cells

The hardware configuration needed to thaw fibroblasts, infectfibroblasts with reprogramming viruses, magnetic sort of reprogrammedcells, and identification of stem cell colonies is composed of threeHamilton STAR liquid handling units (122, 136, 138), two Cytomat 48Cincubators (132), one Cytomat 2C 425 incubator (142), two CyntellectCeligo cytometers (124, 140), Hamilton Capper DeCapper (126), AgilentV-Spin (128), Miltenyi MultiMACS magnetic separation device (136). Theliquid handlers, a Celigo, the Hamilton Capper Decapper and AgilentV-Spin are all connected by a Hamilton Rack Runner robotic rail (130).Each Hamilton STAR is equipped with a Modular Arm for 4/8/12 channelpipetting, 8 pipetting channels, iSWAP plate handler, CO-RE Gripper forplate and lid handling, one or more MultiFlex tilt Module for tiltingplates during media exchanges, one or more Hamilton Heated Shaker 2.0,as well as carrier packages for flexible layout of the liquid handlingplatform with plate and lid parks, pipette stackers, daughter platestackers and troughs for holding media. One of the Hamilton STAR liquidhandlers (122) is also equipped with a 96-well pipetting head. OneCeligo (140) and the Cytomat 2C incubator (142) are connected directlyto one of the Hamilton STARs (138) to facilitate automated cell sorting.The Hamilton STARs are contained within NuAire BSL II biosafety cabinets(144, 146, 148) to maintain a sterile operating environment duringmanipulation of cell culture plates. The remaining components areenclosed in a Hepa filtered hood to maintain a sterile operatingenvironment during transportation of cell culture plates among thedevices. The Cytomat 48C incubator (132) is connected to the othercomponents of the system by the Rack Runner transport rail (130).

To control plate handling on the automated system, MICROLAB STAR VENUSTWO Base Pack 4.3 software controllers (150, 152, 154) with VENUSDynamic Scheduler 5.1 (156) are used in conjunction with individualattached hardware component drivers for all of the Hamilton STARs (122,134, 138), the centrifuge (128), the Capper/decapper (126), the twoCeligos (140, 124), the Rack Runner (130), and Cytomat 24 (132), theCytomat 2C (142), and associated Cytomat transfer stations to integratethe operation of the system. The following methods programmed using theprovided controller software (150, 152, 154) are needed forfunctionality of the system and can be combined in a defined sequence toaccomplish derivation of iPS colonies from fibroblasts:

-   -   1. Load mycoplasma free 6-well biopsy plates (48) onto the STAR        (122) and transfer to the Cytomat incubator (132) under clean        growth conditions (60).    -   2. Confluency check (50) on Celigo (124) and a media exchange on        the STAR (122).    -   3. Passage, harvest (52) and freeze (54, 56) on the STAR (122).    -   4. A thawing method whereby cryotubes containing fibroblasts        (61) are loaded and thawed on the STAR (122), followed by        decapping of tubes (126) and washing of fibroblast, followed by        resuspending cells in plating media and plating fibroblasts on 6        well plates (62) and transferring to Cytomat incubator (132).    -   5. Media change on the STARlet (122) for full media exchange.    -   6. Confluency check on Celigo (124).    -   7. Passaging and seeding of fibroblasts in 24-well plates (64)        on the STARlet (122).    -   8. A method for infection of fibroblasts (64) on the STARlet        (122).    -   9. A method to add a defined volume of media to wells on STAR        (122, 138, 144).    -   10. A method for executing a half media exchange on STAR (122,        138, 144).    -   11. A method for magnetic sorting, dilution and plating (66) on        the STAR (144) attached to the Miltenyi MultiMACS (136) and        Celigo (124).    -   12. A method for a three quarter media exchange on the STAR        (122, 138, 144).    -   13. A method for a executing an immunocytochemical stain on live        colonies followed by automated imaging of the colonies (66)        using a STAR (138) and Celigo (140).    -   14. A method for harvesting, cherry picking and replating        colonies (68) from selected wells on a STAR (138).    -   15. Retrieve plates onto the STARlet (122, 138, 144) from the        Cytomat (132).

Automated System for Expansion, Quality Control, and Freezing ofReprogrammed Cells

The hardware configuration needed to expand reprogrammed Stem CellColonies, generate plates of colonies for quality control assays andgenerate plates and tubes for cryostorage is composed of three HamiltonSTAR liquid handling units (150, 154, 160), Cytomat 24C incubator (172),one Cytomat 2C 425 incubator (174), one Cyntellect Celigo cytometer(166), Hamilton Capper DeCapper (170), Agilent V-Spin (168), and AgilentPlateLoc plate sealer (164). The liquid handlers, a Celigo, the HamiltonCapper Decapper, Agilent V-Spin, and Agilent PlateLoc plate sealer areall connected by a Hamilton Rack Runner robotic rail (162). The HamiltonSTARs and STARlet are equipped with Modular Arms for 4/8/12 channelpipetting, 8 pipetting channels, iSWAP plate handlers, CO-RE Grippersfor plate and lid handling, one or more MultiFlex tilt Modules fortilting plates during media exchanges, one or more Hamilton HeatedShaker 2.0s, as well as a carrier packages for flexible layout of theliquid handling platforms with plate and lid parks, pipette stackers,daughter plate stackers and troughs for holding media. One of the STARs(160) also has a 96 channel Multichannel pipetting head to facilitatemedia exchanges and passaging. The Cytomat 2C and Cytomat 24C incubatorsare connected to the Hamilton STARs by a Hamilton Rack Runner transportrail (162) to facilitate automated media exchanges. The Hamilton STARsare contained within a NuAire BSL II biosafety cabinet (176, 178, 180)to maintain a sterile operating environment during manipulation of cellculture plates. The remaining components are enclosed in a Hepa filteredhood to maintain a sterile operating environment during transportationof cell culture plates among the devices.

To control plate handling on the automated system, MICROLAB STAR VENUSTWO Base Pack 4.3 software controllers (182, 184, 186) with VENUSDynamic Scheduler 5.1 (188) are used in conjunction with individualattached hardware component drivers for the centrifuge, decapper, platesealer, Celigo, and Cytomat incubators and Cytomat transfer station tointegrate the operation of the system. The following methods are neededfor functionality of the system and can be combined in a definedsequence to expand cell cultures in plates for quality control assaysand freezing in plates or cryovials:

-   -   1. A loading method on the STAR (160) to receive plates (68)        from the previous stage into the Cytomat incubator (172).    -   2. Media change on the STAR (150, 154, 160) for full media        exchanges using tilt modules and 8-channel pipetting arms.    -   3. Confluency check on Celigo (166) with associated methods to        transport plates to and from the STARs (150, 154, 160) and        Cytomat incubator (172).    -   4. A method for passaging and seeding of iPSCs in 96-well plates        (68-90) on the STARs (150, 154, 160).    -   5. A method for executing a partial media exchanges on the STARs        (150, 154, 160).    -   6. A method for harvesting, cherry picking and replating        colonies from selected 96-well wells to new 96-well plates (80,        82, 86, 88) on a STAR (150, 154, 160).    -   7. A method for harvesting, cherry picking and replating        colonies from selected 96-well wells to new 24-well plates (90,        92) on a STAR (154).    -   8. A method for harvesting and cherry picking and replating        colonies from selected 24-well wells to new 6-well plates (92,        94) on a STAR (154).    -   9. Passage, harvest and distribute cells in freezing plates (88)        on the STAR (154).    -   10. Passage, harvest and distribute cells in cryotubes (96) on        the STAR (154).    -   11. Retrieve plates onto the STARs (150, 154, 160) from the        Cytomat 24C (172) or Cytomat 2C (174).

Example 2 Production of a Fibroblast Bank for Reprogramming

The first step in the workflow to derive iPSCs from patient samples isto obtain and expand adult cells. This is accomplished, for example, byobtaining a skin punch biopsy or discarded dermal tissue, then isolatingand expanding cultures of fibroblasts from the tissue. In the workflowdescribed in the present Example, this is accomplished by the automatedsystem comprised of Systems 1 and 2. The automated components of System1 and 2 (100-120) and System 3 (122-132, 154, 190) perform the stepsneeded to derive a fibroblast bank stored in cryotubes (61) from patientsamples, including plating of a patient biopsy (2, 22-24), outgrowth andpassaging (4, 26-32) (rolling production on liquid handling robot), QC(6, 34-46) (automated testing for mycoplasma), and automated freezing onthe liquid handling robot (8, 48-56). For example, the workflow anddecision tree for production of fibroblasts from biopsies is dividedinto Quarantine (58) and Clean phases (60). As biopsies enter thefacility, a technician plates biopsies in 6-well plates (22) and logsthe plates into the automated incubator (24) to begin the quarantineworkflow. After biopsies are given time to attach to the plate, theliquid handling robot retrieves the plates from the automated incubatorto feed and check confluency of the outgrowth of adult fibroblasts fromthe plated tissue on an automated microscope (26). The plates arereturned to the incubator and allowed to continue to outgrow (28). Theliquid handler removes the plate from the incubator and exchanges themedia for antibiotic and antimycotic free media (30) to prepare formycoplasma testing. The robot moves the plate to the incubator foranother five days (32). The robot then removes the plate and retrievesmedia to daughter plates for mycoplasma test (34). The daughter platesare moved to the Quarantine Assay system for mycoplasma testing (36). Achoice is then made based on a positive signal from the assay (38). Ifall wells of a 6-well plate fail with a positive assay result (40) theyare discarded. If all wells of a 6-well plate are negative and free ofmycoplasma, they are transferred out of quarantine into the clean growthenvironment provided by Systems 3, 4, 5 (46). If some wells are positiveand some wells are negative, the negative wells are maintained inquarantine (42). The negative wells are passaged (44) to new plates,transferred to the incubator, and the source plates containing positivewells are discarded. These cultures proceed through steps to retest formycoplasma (24-38). Clean cultures are monitored for growth (50),passaged (52) and frozen in cryovials (54, 56, 61).

Production of Stem Cell Arrays

To produce iPSCs, Fibroblasts in cryotubes (61) are plated by theautomated system (10), reprogramming factors are introduced by theautomated system (12), iPSCs are isolated by automated sorting andisolation in System (14), desired clones are selected by the automatedsystem (16), and expanded by the automated system (16), automatedquality checks by the automated system (QC) for pluripotent status bymarker assays and embryoid body assays (18), followed by automatedfreezing and storage of desired cells by the automated system (20).These steps are accomplished on the automated systems 3, 4, 5, 6, 7, and8 (122-192).

For example, the automated iPS derivation process begins when 96 patientand control fibroblast samples in cryotubes (61) are plated inindividual wells of a 6-well plate (62). These are passaged at definedcell number into individual wells of a 24-well plate for infection usingviruses encoding reprogramming factors (64). In the next step,reprogrammed samples are depleted of non-reprogrammed cells by cellsorting or magnetic bead-based enrichment and plated at clonal densityin multiple wells in 96-well plates (66). In this example, 6 wells (66)are identified containing a single clone positive for a pluripotencysurface marker. These clones are cherry picked and consolidated into aminimum number of 96-well plates (68). These clones are seriallypassaged until confluence within a well is achieved for each startingsample (72). Each plates' samples are then replicated onto duplicateplates (74, 76), one plate for a Pluripotency quality control assayneeded to determine pluripotent status of the individual clones (74) andone plate for carrying forward in subsequent passages (76). The platethat is carried forward is passaged again into three plates (78, 80,82). One plate is harvested for QC assay that assesses Karyotype andgenetic diversity (78), one plate (82) is passaged onto v-bottom platesto form embryoid bodies (84) for a QC assay that assessesdifferentiation capability of the iPS clones, and the final plate (80)is carried forward for further expansion. Individual clones that do notpass quality control from previous pluripotency QC assays are notcarried forward as indicated by “X” in the wells in FIGS. 4B2 and 4C(80, 82, 90). Remaining clones are consolidated onto as few plates aspossible until one to three clones remain (86). These clones areexpanded for cryopreservation while attached to the plate (88) orfurther expanded (92, 94) and cryopreserved in cryovials (96).

Embryonic stem cells (ES) are also contemplated to be used with theautomated system of the invention to generate differentiated adultcells. ES cells are derived from the blastocyst of an early stage embryoand have the potential to develop into endoderm, ectoderm, and mesoderm(the three germ layers) (i.e., they are “pluripotent”). In vitro, EScells tend to spontaneously differentiate into various types of tissues,and the control of their direction of differentiation can bechallenging. However, some progress has been achieved in the directeddifferentiation of ES cells to particular types of differentiateddaughter cells. For example, it is now possible to direct thedifferentiation of human ES cells to functional midbrain dopaminergicneurons using defined factors added to the cell cultures at definedstages of their stepwise differentiation (see, e.g., Kriks et al., 2011Nature, Nov. 6. doi: 10.1038/nature10648 (Epub)). As differentiation isnot homogenous, it remains necessary to isolate populations of interestfor further study or manipulation. The process and instrumentationdescribed here could be used to first derive and expand pluripotentembryonic stem cells and also isolate subpopulations of theirdifferentiated derivatives by automated methods including automatedmagnetic cell isolation.

For example, whole human blastocysts can be plated on matrices inmulti-well plates amenable to the automated process. Outgrowths fromthese plated blastocysts could be isolated using the same automatedmagnetic isolation procedures performed by the robotic instrumentationand methods described for the isolation of induced pluripotent stemcells. The resulting human embryonic stem cell lines could be expanded,selected by quality control assays and frozen using the same automatedprocedures described herein.

Further, using pluripotent stem cells, either blastocyst derived orinduced by defined factors or by somatic cell nuclear transfer,differentiated derivatives can be isolated using the described workflowand instrumentation. The differentiated derivatives can be obtained bydirected application of defined factors required to induce a cell fatechange or after spontaneous differentiation. For example, inhibitors ofthe TGF beta pathway can be used to induce neural cell fates frompluripotent stem cells. Neural cells can be subsequently isolated fromnon-neural by magnetic bead immunolabeling of surface antigens, such asNCAM. The described workflow and instrumentation can be used tomagnetically isolate, select, culture and expand differentiated cellslike neurons. This process is also applicable to other differentiatedcell types, like cardiac cells, for which there exist antibodies thatrecognize cell surface antigens specific to the cell type of interest.

Multipotent stems cells are also contemplated to be used with theautomated systems of the invention to generate differentiated adultcells. In particular, mesenchymal stem (MS) cells can be employed togenerate differentiated adult cells using the automated systems of theinvention. MS cells are the formative pluripotent blast orembryonic-like cells found in bone marrow, blood, dermis, and periosteumand placenta that are capable of differentiating into specific types ofmesenchymal or connective tissues including adipose, osseous,cartilaginous, elastic, muscular, and fibrous connective tissues. Thespecific differentiation pathway which these cells enter depends uponvarious influences from mechanical influences and/or endogenousbioactive factors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues. Examplesinclude differentiation of MS cells into differentiated cells with theproperties of chondrocytes for cartilage repair, e.g., see U.S. Pat. No.8,048,673.

Chromosomal Testing

In some aspects, the Nanostring nCounter Plex2 Assay Kit is used totarget the 400 genomic loci, often known to be invariant among thepopulation, allows for integrated molecular karyotype analysis coupledwith “fingerprint” tracking of cell line identity. The molecularkaryotype analysis utilizes an average of 8 probes per chromosome arm toverify genomic stability during the course of cell culture derivationand expansion of iPSC lines. Identity analysis will also be performed onall lines based on 30 common copy number varations (CNVs) of polymorphicloci, which allows for unambiguous identification of individual genomes.

Pluripotency Analysis

In one aspect, surface marker staining is performed to show that cellsare positive for Tra-1-60 surface marker, which is monitored e.g., withthe Celigo automated imager. PSC lines must show a significant level ofthe pluripotency genes. In one example, a probe set of 100 gene makers(described below) was utilized that includes the six markers forpluripotency (Oct4, Klf4, cMyc, Nanog, Lin28, ZFP42, and Sox2). Toperform this analysis a sample of cells was lysed and RNA was harvested.The nCounter Plex2 Assay Kit was used to analyze expression levels inmultiple samples and hundreds of gene targets simultaneously enablingthe high-throughput approach to PSC characterization. As the nCountergene expression assays are quantitative, selection criteria is based onexpression levels falling within a range relative to a control panel ofestablished hESC lines analyzed grown under identical conditions. Linesthat pass pluripotency gene expression criteria will be further expandedand differentiated in vitro in embryoid body (EB) assays.

EB Formation Gene Expression Assay

It has been shown that epigenetic and transcriptional variation iscommon among human pluripotent cell lines and that this variation canhave significant impact on a cell line's utility. In an illustrativeexample, the panels of gene markers includes: 83 different gene markersselected from each of the 3 germ layers (83), 5 retrovirus transgene (4factors with single detection probe, 1 probe), 5 sendai transgenes (4factors+vector only, 5), Oct4, Klf4, cMyc, Nanog, Lin28, ZFP42(pluripotency, Sox2 is in germlayer group, 6 probes), sex markers (SRY,XIST (2)—donor sex must match or lines will be rejected), andhousekeeping genes, ACTB, POLR2A, ALAS1 (3 probes).

hPSC Line Expansion and Storage

Automated Expansion

Cell lines are expanded through plating of the initial cells into 2separate wells of a 6-well plates then placing them within a CO2incubator and allowing them to grow up to a maximum of 95% confluence.

Storage

The vials are first placed within the SAM −80 freezer to perform theinitial slow cool. This system has automated monitoring of temperatureand logs of time the system is accessed.

Next, the vials are placed in LN2 for long-term storage. Quality controlfor monitoring is detailed later in this proposal. Each vial isindividually marked with a unique 2D barcode and inventory is trackedwithin the LIMS.

hPSC Line Characterization

iPSC and EB gene expression analysis-Set of probes covering lineagedifferentiation assay scorecard (100 genes) to monitor germ layerdifferentiation in EB assays, pluripotency markers, sex markers andtransgene expression

Freeze-Thaw Analysis

Cells are counted following recovery and plated in one well of a 6-wellplate. Colonies are photographed on the first day of appearance and then5 days later, colonies must display a doubling time no larger than 36hours.

Surface Marker Analysis

Perform surface marker analysis using automated system using highcontent imaging of Tra-1-60 staining using the Celigo automated imager.

iPSC and EB Gene Expression Analysis

Pluripotency gene expression—iPSC clones must show a significant levelof the pluripotency genes. A probe set of 100 gene makers (describedbelow) was used that includes the six markers for pluripotency (Oct4,Klf4, cMyc, Nanog, Lin28, ZFP42, and Sox2). To perform this analysis asample of cells is lysed for each of the selected clones and RNA isharvested. The nCounter Plex2 Assay Kit was used to analyze expressionlevels in multiple samples and hundreds of gene targets simultaneouslyenabling the high-throughput approach to iPSC characterization. As thenCounter gene expression assays are quantitative, selection criteria isbased on expression levels falling within a range relative to a controlpanel of established hESC lines analyzed grown under identicalconditions. Selected clones that pass pluripotency gene expressioncriteria will be further expanded and differentiated in vitro inembryoid body assays.

EB formation gene expression assay—In order to firmly establish thenature and magnitude of epigenetic variation that exists among humanpluripotent stem cell lines, three genomic assays were applied to 20established embryonic stem cell (ESC) lines and 12 iPSC lines that wererecently derived and functionally characterized. As a step towardlowering the experimental burden of comprehensive cell linecharacterization, and to improve the accuracy over standard existingassays, all of the data from these studies are combined using the threegenomic assays into a bioinformatics scorecard, which enableshigh-throughput prediction of the quality and utility of any pluripotentcell line. This scorecard was used to analyze gene expression data fromthe EBs formed from each clone of the iPSC lines. To testdifferentiation potential, the automated system was used to generate EBsin 96-well v-bottom plates and ends in RNA harvest for NanostringnCounter Plex2 Assay Kit. The score card comprised 83 different genemarkers selected from each of the 3 germ layers (83), 5 retrovirustransgenes (4 factors with single detection probe, 1 probe), 5 sendaitransgenes (4 factors+vector only, 5), Oct4, Klf4, cMyc, Nanog, Lin28,ZFP42 (pluripotency, Sox2 is in germlayer group, 6 probes), sex markers(SRY, XIST (2)), and housekeeping genes (ACTB, POLR2A, ALAS1 (3 probes).

Karyotype and Identity Analysis

Prior to accepting a line and at the end of each expansion, theNanostring nCounter Plex2 Assay Kit was used to target the 400 genomicloci allowed for integrated molecular karyotype analysis coupled with“fingerprint” tracking of cell line identity. The molecular karyotypeanalysis utilizes an average of 8 probes per chromosome arm to verifygenomic stability during the course of cell culture derivation andexpansion of iPSC lines. The “fingerprint” identity tracking analysiswill rely on a combinatorial signature based on 30 common copy numbervariations (CNVs) of polymorphic loci, which allows for unambiguousidentification of individual genomes. Additionally to avoidmisidentification, tissue donors known to be relatives will not beprocessed in the same batch, as it is theoretically possible they willhave similar CNVs. The data from the identity analysis will becross-referenced with the initial CNV data to ensure that the LIMSsystem properly tracked all cell lines.

Freeze-Thaw Analysis

Freeze-Thaw Analysis: one vial is thawed After cryopreservation. Cellsare counted following recovery and plated in one well of a 6-well plate.Cultures are observed daily. Colonies are photographed on the first dayof appearance and then 5 days later. Colonies must at least double indiameter within 5 days after first observation.

Automated Biopsy Outgrowth Tracking

Using the invention system, one can track the outgrowth of biopsies aswell as other tissue sources by automated and traceable image analysis.As shown in FIGS. 6A-6D, images and growth rates are tracked during theproduction process. In FIG. 6A, biopsies or discarded tissue are platedin multiple wells of a 6-well dish and maintained by an automated systemthat feeds, images, passages, and freezes fibroblast outgrowths.Examples of the image analysis interface is shown for a typical sample.A single plate is used per donated sample to minimize crosscontamination. (FIG. 6B) Cell numbers are extrapolated from confluencemeasurements based on linear regression from a standard curve generatedindependently. (FIGS. 6C and 6D) An example of cell counts for a typicalbiopsy outgrowth maintained on the automated system. Extrapolated cellnumbers per patient sample are plotted for each well independently (FIG.6C) allowing calculation of total output from the sample (FIG. 6D).

FIGS. 7A-7D show FACS analyses and graphs showing automated iPSCreprogramming. Expression levels of pluripotent surface markers onreprogrammed human fibroblasts were followed over a 3 week period toobserve reprogramming kinetics and determine optimal time points atwhich to isolate defined cell populations. (FIG. 7A) FACS gating schemeused for analysis. (FIG. 7B) A substantial proportion of cellsco-expressing traditional pluripotency surface markers SSEA4 & TRA-1-60retain the fibroblast marker CD13 at all timepoints during reprogrammingusing either retroviral or Sendai vectors to introduce reprogrammingfactors Oct4, Sox2, Klf4 and c-Myc. Box plots indicating aggregated datafrom 131 experiments (Retrovirus, n=66, Sendai virus, n=65) are shown.While Sendai mediated reprogramming produces more SSEA4/TRA-1-60 doublepositive cells, (FIG. 7C) there is a delay in elimination of CD13 fromthe surface. (FIG. 7D) Example staining pattern of a patient cell linereprogrammed using Sendai/Cytotune system on the automated system. Atboth 7 and 13 dpi, more than half of SSEA4/TRA-1-60 double positivecells have lost CD13. Additionally, at both timepoints assayed, CD13negative/Nanog positive cells are present in this fraction, suggestingthese can be isolated by negative selection against CD13.

FIGS. 8A-8C show FACs pre-sort analyses and a part of the automatedsystem to demonstrate enrichment and clone selection of iPSCs. (FIG. 8A)Non-reprogrammed cell populations can be depleted from cultures of iPSCsby negative selection by a fibroblast marker. This strategy leaves iPSCsuntouched. In the example, fibroblasts are efficiently removed from theculture containing 2% established iPSCs leaving TRA-1-60 positive iPSCsuntouched. (FIG. 8B) A Miltenyi MultiMACS system integrated intoHamilton liquid handler can sort 24 samples in parallel. (FIG. 8C) Anexample colony of newly derived iPSCs derived by negative selectionusing anti-fibroblast antibody conjugated magnetic beads on theMultiMACS system. Phase contrast, nuclear stain by Sytox, surface markerstain by TRA-1-60 and nuclear Nanog staining. The iPS enriched fractionfrom the anti-fibroblast magnetic negative selection step is plated on96-well imaging plates at limiting dilution. These plates are screenedusing live-cell staining for the pluripotency surface marker TRA-1-60 orTRA-1-81. Wells with TRA-1-60 positive iPSCs are identified by automatedimage analysis using the Celigo software capable of single colonyconfirmation. Wells that meet both criteria of containing a singlecolony that is positive for the surface marker are selecting forpassaging and expansion and QC. Colonies produced by automated Sendaiinfection of adult fibroblasts.

iPSC induction has also been demonstrated by automated transfection ofmodified mRNA. iPSC colonies from BJ fibroblasts were efficientlyrecovered after 10 days of automated delivery of a transfection mixcontaining modified mRNA. After an additional two days culture, the samewell was stained with TRA-1-60 to identify undifferentiated cells. iPSCsin the well demonstrate that these are undifferentiated iPSCs. iPSCcolonies isolated by purification away from non-reprogrammed cells usingmagnetic bead depletion on the automated system were efficientlyrecovered.

High throughput scorecard assays for gene expression have beengenerated. The first stage of a quality control screen uses a panel ofpluripotency differentiation and transgene markers to choose an initialset of three clones. FIG. 9A shows transcript counts after normalizationto HK gene expression for two HESC lines, Sendai positive control,fibroblast negative control, and iPS lines derived by FACS sortingassayed at passage 5 and 10. All assays are run relative to a panel ofnormal HESC and iPS lines maintained under similar conditions. Not shownwas an example image of an Embryoid body generated on the system in96-well V-bottom plates. The arrow points to the EB. FIG. 9B illustratesthe second stage of a quality control screen uses an additional 83 germlayer/lineage markers to monitor differentiation capability in embryoidbody assays. Single EBs are generated and pooled to generate RNA forexpression analysis of germ layer markers in the embryoid body scorecardassay. Shown is a cluster dendrogram analysis of gene expression in EBscollected from nine different embryonic stem cells lines. Afternormalization, data generated from direct lysis of six EBs comparesfavorably to data generated from total RNA extracted and purified fromEBs prepared from bulk culture.

FIGS. 10A-10B demonstrate high throughput karyotyping of iPSCs based onNanostring nCounter assays for CNVs. FIG. 10A is an example of thenCounter Karyotype assay on BC1 iPSCs; FIG. 10B is an example of thenCounter Karyotype assay on 1016 fibroblasts with partial gain and lossof chromosome arms. Comparison to Affymetrix SNP 6.0 chip datademonstrating copy number gains on a portion of the q arm of Chr1 (toptrack, 1q21.2-1q43) and loss of part of the long arm of Chr6 (bottomtrack, 6q16.3-6q26).

Example 3 Improved Methods for Reprogramming Human Dermal Fibroblasts

The work described in the present Example, including the associatedfigures, tables and drawings, was published by one or more of theinventors of the present application and others. The citation for thepublication is Kahler et al. (2013) “Improved Methods for ReprogrammingHuman Dermal Fibroblasts Using Fluorescence Activated Cell Sorting,”PLoS ONE 8(3): e59867, published Mar. 29, 2013, and the entire contentsof Kahler et al. 2013, including its figures, supplemental figures, andsupplemental tables, are incorporated herein by reference in theirentirety.

Current methods to derive induced pluripotent stem cell (iPSC) linesfrom human dermal fibroblasts by viral infection rely on expensive andlengthy protocols. One major factor contributing to the time required toderive lines is the ability of researchers to identify fullyreprogrammed unique candidate clones from a mixed cell populationcontaining transformed or partially reprogrammed cells and fibroblastsat an early time point post infection. Failure to select high qualitycolonies early in the derivation process results in cell lines thatrequire increased maintenance and unreliable experimental outcomes.Here, an improved method is described for the derivation of iPSC linesusing fluorescence activated cell sorting (FACS) to isolate single cellsexpressing the cell surface marker signature CD13NEGSSEA4POSTra-1-60POSon day 7-10 after infection. This technique prospectively isolates fullyreprogrammed iPSCs, and depletes both parental and “contaminating”partially reprogrammed fibroblasts, thereby substantially reducing thetime and reagents required to generate iPSC lines without the use ofdefined small molecule cocktails. FACS derived iPSC lines express commonmarkers of pluripotency, and possess spontaneous differentiationpotential in vitro and in vivo. To demonstrate the suitability of FACSfor high-throughput iPSC generation, 228 individual iPSC lines werederived using either integrating (retroviral) or non-integrating (Sendaivirus) reprogramming vectors and performed extensive characterization ona subset of those lines. The iPSC lines used in this study were derivedfrom 76 unique samples from a variety of tissue sources, including freshor frozen fibroblasts generated from biopsies harvested from healthy ordisease patients.

INTRODUCTION

The discovery that differentiated somatic cells could be reprogrammed toan embryonic stem cell-like state by forced expression of fourtranscription factors (Oct4, Klf4, Sox2, cMyc) has revolutionized thestem cell field [1]. Reprogrammed, or induced pluripotent stem cells(iPSCs), show remarkable similarities to embryonic stem cells (ESCs) andhold great promise for in vitro disease modeling, drug discovery, andtherapeutic interventions because they provide a potentially unlimitedsource of differentiated cells from individuals with specific diseases[2], [3], [4], [5], [6].

However, initial derivation of stable iPSC clones by viral transductionof dermal fibroblasts is a slow (4-6 weeks) and inefficient (<0.01% oftotal fibroblasts) process. Current methods of identifying colonies ofbona fide iPSCs early in the reprogramming process (2-3 weekspost-infection) utilize light microscopy and manual isolation ofcandidate colonies, which requires training and expertise in advancedcell culture techniques. To enable future clinical applicationsrequiring de novo iPSC derivation, there remains a need for standardizedand validated methods for identifying, isolating and purifyingreprogrammed cells.

Previous imaging studies based on tracking of cell-of-origin suggestthat early events occur during defined factor reprogramming, including achange in cell proliferation rates and morphology [7], downregulation ofCD13, a marker of mesenchymal cells including fibroblasts [8], as wellas upregulation of the cell surface markers of pluripotency SSEA4 andTRA-1-60 [9]. These studies demonstrate that both partially and fullyreprogrammed iPSCs can be identified by combined use of surfaceexpression of multiple markers. Recently, a method of enrichingreprogrammed fibroblasts by fluorescence activated cell sorting (FACS)for cells with dual expression of the pluripotency surface markers SSEA4and TRA-1-81 arising late during reprogramming was described [10]. Whilea step forward, this method relies heavily on the use of a defined smallmolecule cocktail, and multiple rounds of sorting and extensivescreening to identify fully reprogrammed clones. This suggests thatpluripotency markers alone are not sufficient to purify fullyreprogrammed iPSCs. Additionally, it is likely that the high variabilityamong clones seen within this population is compounded by the use ofintegrating vectors to deliver the reprogramming factors. Here, resultsconfirm that throughout the reprogramming process a significantproportion of SSEA4POSTra-1-60POS cells retain the fibroblast surfacemarker, CD13. Through the use of negative selection against CD13, fullyreprogrammed iPSCs were purified from partially reprogrammed cells andparental fibroblasts by FACS. This method removes contaminating cells atan early stage and can be used with a variety of cell populationsincluding: cells reprogrammed with DNA-integrating or non-integratingviruses; fibroblasts harvested from healthy or specific diseasepatients. Using this method, 228 iPSC lines have been generated andcharacterized from 76 fibroblast lines obtained from multiple sourcesincluding fresh biopsies, frozen stocks, and cell line repositories.

Materials and Methods Fibroblast Cell Culture

Explants of 3-mm dermal biopsies were minced and placed in a 60-mmtissue culture dish under a sterile coverslip held down by sterilizedsilicon grease. Fibroblast medium [Dulbecco's modified Eagle's medium(DMEM), supplemented with 10% fetal bovine serum, Glutamax™, andpenicillin/streptomycin (Invitrogen, Carlsbad, Calif.)] was added, anddishes incubated at 37° C. in a humidified 5% CO2 atmosphere with mediaexchange every 5 days. Fibroblast outgrowths were harvested bytrypsinization, expanded in a T25 flask in fibroblast medium, andallowed to reach ˜90% confluence prior to freezing or splitting forreprogramming as described below. For reprogramming, fibroblasts wereused within the first three passages from biopsy or within one passageafter a thawing. All parent fibroblast and reprogrammed lines weresubjected to cytogenetic analysis (Cell Line Genetics) and for DNAfingerprinting by short tandem repeat (STR) analysis.

Fibroblast Reprogramming

Fibroblasts were reprogrammed using high titer stocks of vesicularstomatitis virus G (VSVG)-coated retroviruses containing Oct4, Sox2,cMyc, and Klf4 (Harvard Gene Therapy Initiative at Harvard MedicalSchool) as previously described [12], or the non-integratingCytoTune™—Sendai viral vector kit (Life Technologies, A13780).Fibroblasts reprogrammed with retroviruses were infected at 1×104cells/well in 1 ml of human ESC medium (HUESM) [Knockout™ DMEMsupplemented with 20% Knockout™ serum replacement (Invitrogen), 10 ng/mlbFGF (Invitrogen), nonessential amino acids (Invitrogen),β-mercaptoethanol (Invitrogen), L-glutamine, and penicillin/streptomycin(Invitrogen)]. The medium was exchanged on day 2 to HUESM containingALK5 inhibitor SB431542 (2 μM; Stemgent), MEK inhibitor PD0325901 (0.5μM; Stemgent), and ROCK inhibitor [13] Thiazovivin (0.5 μM; Stemgent)]and changed daily thereafter. For Sendai virus mediated reprogramming,5×105 fibroblasts were infected in fibroblast medium at a multiplicityof infection of 3 (MOI3) for two days with daily (HUESM) mediaexchanges. At day 7-10 days post-infection by retro or Sendai viralprotocols, cells were subjected to FACS analysis or passaged onto feedercells by enzymatic dissociation using Dispase (GIBCO) and/or Accutase(Sigma-Aldrich) then passaged onto γ-irradiated murine embryonicfibroblasts (MEFs; Globalstem) or Matrigel™ (BD Biosciences) coatedplates in HUESM at 2×103 cells/cm2.

Fluorescent Activated Cell Sorting of Reprogrammed Fibroblasts

Cells enzymatically harvested as described above were filtered through a35 μm cell strainer (BD biosciences) to obtain a single cell suspensionprior to resuspension in 100 μl of a sterile iPSC staining buffer [DPBScontaining 0.5% bovine serum albumin fraction V (BSA; Invitrogen), 100U/ml penicillin/streptomycin (Invitrogen), 2 mM EDTA (Invitrogen), and20 mM glucose (Sigma)]. A cocktail of fluorescence-conjugated antibodies[1 μl each anti-CD13 (555394) anti-SSEA4 (560219) and anti-Tra-1-60(560173), obtained from BD, CA] was added to the cells and incubated atroom temperature (RT) for 15 minutes shielded from light. Stained cellswere washed once with iPSC staining buffer and sorted immediately on a 5laser BD Biosciences ARIA-IIu™ SOU Cell Sorter using “gentle FACS”sorting conditions based on the work of Pruszak et al. (100 μm ceramicnozzle, 20 psi) [14]. Some experiments included antibodies against SSEA3(BD, 560237), or CD326 (BD, 347200) in the cocktail to confirm thepluripotent status of the reprogrammed cells. Target cell populationswere sorted directly onto MEF layers (ARIA plate holder at 37° C.) atbetween 2×103 and 5×104 cells/well in a 6-well plate containing HUESMplus 20 μM Y-27632 (ROCK inhibitor; Calbiochem). Two days after sorting,the ROCK inhibitor was removed from the medium and replaced withSB431542 (2 PD0325901 (0.5 and Thiazovivin (0.5 μM) [13] with dailymedia exchange. Several individual colonies were picked 7-10 days aftersorting and expanded for characterization.

Quantitative RT-PCR

Total RNA was isolated from duplicate or triplicate cell samples usingthe RNeasy kit (QIAGEN, 74136). cDNA synthesis was performed on 1 μg RNAwith SuperScript™ III First-Strand system (Invitrogen 18080-051) andoligo(dT) primers. The cDNA was diluted to a final volume of 200 μl and1 μl was added to 500 nM of the forward and reverse primers in a finalvolume of 10 μl per PCR reaction. Quantitative real-time PCR wasperformed using the GoTaq® SYBR Green Master kit (Promega, A6001) andMx3000p QPCR system (Stratagene). Primer sequences are provided in Table3, below.

TABLE 3 Quantitative Real-Time PCR Primers (S1) FORWARD PRIMERREVERSE PRIMER GENE 5′-3′ 5′-3′ Oct 4 CCCCAGGGCCCCATT GGCACAAACTCCAGG(endo- TTGGTACC TTTTC genous) (SEQ ID NO: 1) (SEQ ID NO: 2) Sox2ACACTGCCCCTCTCA GGGTTTTCTCCATGC (endo- CACAT TGTTTCT genous)(SEQ ID NO: 3) (SEQ ID NO: 4) Klf4 ACCCACACAGGTGAG GTTGGGAACTTGACC(endo- AAACCTT ATGATTG genous) (SEQ ID NO: 5) (SEQ ID NO: 6) C-MycAGCAGAGGAGCAAAA CCAAAGTCCAATTTG (endo- GCTCATT AGGCAGT genous)(SEQ ID NO: 7) (SEQ ID NO: 8) Oct4 CCCCAGGGCCCCATT AACCTACAGGTGGGG(trans- TTGGTACC TCTTTCA gene) (SEQ ID NO: 9) (SEQ ID NO: 10) Sox2ACACTGCCCCTCTCA AACCTACAGGTGGGG (trans- CACAT TCTTTCA gene)(SEQ ID NO: 11) (SEQ ID NO: 12) Klf4 GACCACCTCGCCTTA AACCTACAGGTGGGG(trans- CACAT TCTTTCA gene) (SEQ ID NO: 13) (SEQ ID NO: 14) C-MycAGCAGAGGAGCAAAA AACCTACAGGTGGGG (trans- GCTCATT TCTTTCA gene)(SEQ ID NO: 15) (SEQ ID NO: 16) B2M TAGCTGTGCTCGGGC TCTCTGCTGGATGAC TACTGCG (SEQ ID NO: 17) (SEQ ID NO: 18)

Southern Blotting

Probes for human Oct4, Sox2, and KLF4 were generated by PCR using thedigoxigenin (DIG) probe synthesis kit (Roche) and Southern blotting wasperformed using DIG System detection reagents (Roche). Genomic DNA wasisolated from human ESCs, parent fibroblast cells, and iPSCs using theQiagen DNA Mini kit. DNA samples (5-10 μg) were digested overnight withBglII to generate a single cut in the integrated viral backbone of eachtransgene, and digests were resolved on a 0.8% agarose gel (withoutethidium bromide), which was then denatured with 0.5% NaOH andneutralized. The gel was transferred to a nylon membrane by overnightcapillary transfer. Wet membranes were crosslinked with 120 mJ UV(HL-2000 Hybrilinker, UVP) and allowed to dry. Membranes werepre-hybridized with DIG Easy Hyb buffer for at least 1 h at 55° C., thenincubated with the appropriate probe overnight at 55° C. Membranes werewashed thoroughly using the DIG Wash and Block kit, blocked for at least1 h, and incubated with anti-DIG antibody for 30 min. Membranes werethen washed and treated with CDP-Star reagent to detect DIG-incorporatedbands. Blots were stripped and re-probed according to the manufacturer'sinstructions. Probe sequences are provided in Table 4, below.

TABLE 4 Southern Blot Primers (S2) FORWARD PRIMER REVERSE PRIMER GENE5′-3′ 5′-3′ Oct 4 GAGAAGGAGAAGCT GTGAAGTGAGGGCT (endo- GGAGCA CCCATAgenous) (SEQ ID NO: 19) (SEQ ID NO: 20) Sox2 AGAACCCCAAGATGTGGAGTGGGAGGAA (endo- CACAAC GAGGTA genous) (SEQ ID NO: 21)(SEQ ID NO: 22) Klf4 ACCTGGCGAGTCTG TCTTCATGTGTAAGGCG (endo- ACATGGAGGTGG genous) (SEQ ID NO: 23) (SEQ ID NO: 24)NanoString nCounter Assay

Total RNA was isolated from each iPSC clone between passage 10 and 15using the RNeasy kit (Qiagen). A 100-ng sample of RNA was then profiledusing the NanoString nCounter system (NanoString, Seattle, Wash.) usingone of two custom-designed codesets. The pluripotency codeset contains25 probes for detection of the Sendai and retroviral transgenes,pluripotency and spontaneous differentiation markers, and housekeepinggenes (Table 5). The lineage codeset is derived from a previous study[15] and contains 85 probes for the three germ layers in addition toprobes for retroviral and Sendai transgenes, and housekeeping genes(Table 6). RNA from a retrovirus-positive or Sendai-positive controlline, a fibroblast line (1043), and two human ESC lines (HUES42 andHues16) was included in each run. Data were analyzed using the nSolverAnalysis Software v1.0 (NanoString) and plotted using Prism (GraphpadSoftware, La Jolla, Calif.). Data quality control and normalization togeometric mean for both internal positive controls, and subsequently forhousekeeping genes, was performed in the nSolver analysis software.

TABLE 5 Pluripotency Codeset (S3) Sendai Pluripotency SpontaneousRetroviral transgenes Markers Differentiation Fibroblasts HousekeepingtOct4 S-tOct4 POU5F1 SOX17 ANPEP ACTB (OCT4) (CD13) tSox2 S-tKlf4 SOX2AFP POLR2A tKlf4 S-tC-myc KLF4 NR2F2 ALAS1 tC-Myc S-tSox2 MYC SeV LIN28NANOG ZFP42

TABLE 6 Lineage Codeset (S4) Mesoderm Ectoderm Endoderm RetroviralSendai Pluripotent Other Housekeeping ABCG2 ABCG2 APOE tOct4 tOct4POU5F1 SRY ACTB ADIPOQ APOE CD44 tSox2 tSox2 NANOG XIST POLR2A ANPEPCD44 CDH2 tKlf4 tKlf4 ZFP42 ALAS1 CD34 CDH2 CDX2 tC-Myc tC-Myc CD36CRABP2 CTNNB1 SeV CD4 EN1 FOXA2 CD44 FAS GATA4 CDH1 FGFR2 GATA6 CDH2FUT4 GCG CDH5 GATA2 HNF1A CEACAM1 GATA3 HNF1B DLL1 HAND1 ISL1 FUT4 ICAM1ITGA6 GATA3 ITGA4 ITGB1 GATA4 ITGA6 NEUROG3 HHEX ITGB1 NKX2-5 ICAM1 MAP2PAX6 INHBA MAPT PDX1 ITGA4 MCAM SLC2A2 ITGA6 MNX1 SST ITGAL NCAM1 SYPITGAM NEFL THY1 ITGAV NES ITGAX NEUROG3 ITGB1 NGFR ITGB3 NOG KDR NOTCH1KIT OTX2 LEF1 PAX3 MCAM PAX6 MME PAX7 MYOD1 PDGFRA MYOG SNAI2 NCAM1SOX10 NES SOX2 NGFR SOX9 NOTCH1 SYP PECAM1 TDGF1 SDC1 TH SPI1 THY1 SRFSTAT3 T THY1 TNFRSF1A TWIST1

Embryoid Body Formation

Embryoid bodies (EB) were formed by placing clumps of iPSCs in 96-wellnon-tissue culture treated V-bottom plates (Evergreen 222-8031-01V)containing HUESM. After 5 weeks of culture, EBs were harvested, fixed in4% paraformaldehyde (PFA) for 30 min at RT and processed in 15% and 30%sucrose solutions for one day each prior embedding in O.C.T., freezing,sectioning into 10 μm slices and mounting on glass slides. EB sectionswere immunostained with antibodies against the markers shown in Table 7,below. Briefly, EB sections were incubated with blocking solution 10%donkey serum in PBST (PBS with 0.1% Triton-100) for 1 h at RT, followedby an overnight incubation at 4° C. with primary antibodies. Afterwashing three times with PBST, sections were incubated for 1 h at RTwith appropriate secondary antibodies obtained from Molecular Probes.Finally, sections were washed and counterstained with DAPI (1:1000 inPBS) for 15 min at RT.

TABLE 7 Primary Antibodies for Immunofluorescence (S5) Antibody CompanyCatalog # Dilution Oct4 Stemgent 09-0023 1:250 Sox2 Stemgent 09-00241:250 Tra-1-60 Millipore MAB4381 1:250 SSEA4 R&D Systems MAB1435 1:250Nanog R&D Systems AF1997 1:250 SSEA3 R&D Systems MAB1434 1:250 SmoothMuscle DAKO M085101 1:500 Alpha-1-Fetoprotein DAKO A0502 1:500 TUJ1Covance MMS-435P 1:500 Nestin Millipore AB5922 1:500 MAP2 Abcam ab324541:500

Teratoma Assay

Manually (1023_C) and FACS-derived (1023 D2) cells were dissociatedusing Dispase (Gibco 17105-041) for 15 minutes at 37° C. to producesmall clumps containing approximately 100-200 iPSCs/clump. The clumpswere suspended in 100 μl of HUESM containing 100 μl Matrigel™ (BDBiosciences) and injected subcutaneously into NOD-SCID Il2rg-null mice(Jackson Laboratory) following an intraperitoneal injection of carprofen(Pfizer) at 5 mg/kg. Teratomas were allowed to grow for 6-8 weeks,isolated by dissection and fixed in 4% PFA overnight at 4° C. Fixedtissues were embedded in paraffin, sectioned at 10 μm and stained withhematoxylin and eosin (H&E).

Results

FACS Derivation of iPSCs

Previous studies have demonstrated downregulation of the humanfibroblast marker CD13 [8], and upregulation of the pluripotency markersSSEA4 and TRA-1-60 occurs during reprogramming [9]. These studiessuggest that isolation of fully reprogrammed iPSCs during early stagesof reprogramming may be accomplished by FACS using a combination ofpositive and negative surface markers. While current sorting strategiesfor purification of pluripotent cells rely on positive selection [7], itis possible that a significant proportion of clones isolated using thismethod may not be fully reprogrammed. To test this hypothesis, first theconditions for survival of live-cell sorted, fully reprogrammed cellswas optimized by examining the expression levels of three surfacemarkers in a manually derived, early passage clone (p4) of an iPSC line(1018) cultured on MEFs.

Populations of cells expressing all three markers were found in theculture suggesting a heterogeneous mixture of cells containing parentaland partially reprogrammed fibroblasts FIG. 11A. Then theCD13NEGSSEA4POSTra-1-60POS (Tra-1-60POS) population was sorted and, as acontrol, the CD13NEGSSEA4POSTra-1-60NEG (Tra-1-60NEG) population wassorted to approximately 70% purity directly into one well of a 6 wellplate containing MEFs in the presence of ROCK inhibitor Y-27632. Thesorted populations were maintained for 20 days on MEFs without ROCKinhibitor or removal of differentiated cells or splitting prior toreanalysis by flow cytometry (FCM). At 20 days post-sorting (dps), thecultures originating from the enriched Tra-1-60POS population containedfewer Tra-1-60NEG differentiated cells and no detectable CD13POSparental fibroblasts. The Tra-1-60POS population was present in thesecultures at approximately double the proportion found in the Tra-1-60NEGenriched culture (30% vs. 14%). Conversely, at 20 dps the cultureoriginating from the Tra-1-60NEG enriched population contained aTra-1-60POS population at a similar proportion to the originally sortedculture (18% T vs. 14% T). However, these cultures also contained ahigher proportion of differentiated or transformed cells(CD13NEGSSEA4NEGTra-1-60NEG) as well as adult fibroblasts (CD13POS)suggesting that these FACS conditions allow for purification of fullyreprogrammed cells from contaminating cell types.

To confirm this strategy, adult skin fibroblasts at 8 days postinfection (dpi) were sorted based on the CD13NEGSSEA4POSTra-1-60POSsurface marker profile. As a control, the CD13NEGSSEA4POS populationthat contained two subpopulations of cells expressing either Tra-1-60POSor Tra-1-60NEG was isolated in parallel. 5,000 or 10,000 cells from eachpopulation were sorted directly into MEF-containing plates, andmonitored for the formation of colonies. Small but distinct colonieswere evident in both sorted populations as early as 3 days post sort(dps), with the CD13NEGSSEA4POSTra-1-60POS population producing largerand more abundant colonies than the CD13NEGSSEA4POS population (3 dps,11 dpi; FIG. 11B). Following an additional 2 weeks of expansion withoutmanual removal of differentiated cells, wells containing theCD13NEGSSEA4POS population had become overgrown with transformed anddifferentiated cells, whereas wells containing the sortedCD13NEGSSEA4POSTra-1-60POS cells contained large, well-separatedcolonies with few differentiated cells between the colonies and lackedcells with transformed morphology (17 dps, 25 dpi; FIG. 11B). A 3-4 foldincrease was observed in the number of colonies present in wellscontaining the sorted CD13NEGSSEA4POSTra-1-60POS cells compared to thesorted CD13NEGSSEA4POS cells FIG. 11C.

CD13 expression was then analyzed within the SSEA4POSTra-1-60POSpopulation of reprogrammed fibroblasts at 7 dpi. Roughly one quarter ofthe SSEA4POSTra-1-60POS population expressed CD13 indicating thepresence of a heterogeneous population of fully reprogrammed,transformed, or transitioning cells (23% CD13POS, 66% CD13−; FIG. 11D),some of which expressed both Nanog and CD13 FIG. 11E.

Because surface marker expression during reprogramming is dynamic, theearliest time point was first identified at which to enrich fullyreprogrammed iPSCs. Time course analysis conducted by flow cytometryfollowing retroviral reprograming suggested that SSEA4POSTra-1-60POScells were detectable as early as 7 dpi, and their proportion increasedup to 21 dpi, then remained constant as marker negative cells outgrewthe reprogrammed cells Table 3. The SSEA4POSTra-1-60POS population alsoexpressed the SSEA3 and CD326 pluripotency markers [16], [17], [18],[19]. SSEA4POSCD13POS cells appeared by 7 dpi and while most of thispopulation disappeared by 14 dpi, a proportion remained in the culture.To test whether this timing was consistent among different skin samples,cultures derived from a foreskin fibroblast line (0825), a healthy adultcontrol fibroblast line (1023), and a fibroblast line from a subjectwith type I diabetes (1018) were analyzed for up to 21 dpi. The threefibroblast cell lines showed a consistent emergence of pluripotentsurface markers with SSEA4POSTra-1-60POS cells present at low numbers at7 dpi (D7, 0.3%-0.4% FIG. 12A), increasing in proportion at 14 dpi (D14,1.4%-2.2%), and decreasing by day 21 as other cells overtook the culture(D21, 0.7%), suggesting a consistent appearance of potentially earlyreprogrammed cells between 7 and 14 dpi. However, at early time points,the majority of SSEA4POSTra-1-60POS cells also expressed CD13 (D7,98%-100%). The proportion of CD13POSSSEA4POSTra-1-60POS decreasedapproximately half by day 14 post infection (D14, 37%-54%), suggestingloss of this fibroblast marker on cells undergoing reprogramming.Interestingly, the CD13POSSSEA4POSTra-1-60POS population increased againby day 21, suggesting that this population was expanding or that CD13NEGcells were lost from the culture.

Based on these results, the sorting strategy shown in FIG. 12B wasdeveloped which omits the contaminating partially reprogrammedCD13POSSSEA4POS population by selecting the highest Tra-1-60POSexpressing cells within the CD13NEGSSEA4POS population. Using thisstrategy FACS was used to derive 228 individual iPSC lines from over 75fresh or frozen fibroblast lines generated from biopsies harvested fromhealthy or disease patients using either integrating (retroviral) ornon-integrating (Sendai virus) reprogramming vectors and extensivecharacterization was performed on a subset of those lines which isdescribed in the data that follows. The first 2 weeks of thereprogramming process was further characterized on 128 FACS derived iPSClines using the analysis structure shown in FIG. 11D. As shown in FIG.12C, a higher percentage of SSEA4POSTra-1-60POS cells were generated inSendai infections compared to retroviral infections over the entire timecourse. However, Sendai infections demonstrated a delayed reduction inthe proportion of CD13POSSSEA4POSTra-1-60POS cells FIG. 12D. By thesecond week of induction, the proportion of the CD13POS populationbetween the cultures was similar.

FACS Derived Lines are Pluripotent

To further characterize this defined selection strategy, the phenotypeand function of the FACS-derived iPSC clones were compared to manuallypicked clones. First, the 0825, 1018, and 1023 fibroblast lines shown inFIG. 12A reprogrammed using the 4-factor retroviral protocol weresubjected to either FACS derivation at 7 dpi or standard pickingtechniques. For each method, one clone from each line was randomlyselected for expansion and further characterization by a standardbattery of assays, including karyotypic analysis, DNA fingerprint,pluripotent surface marker expression, qRT-PCR, and Embryoid body (EB)and teratoma formation. All fibroblasts and reprogrammed iPSC linesdisplayed normal karyotypes, and had DNA fingerprints matching theparental fibroblast line (Table 4). Clones from the 0825 foreskinfibroblast line had a DNA fingerprint that matched a major subpopulationin the parental fibroblasts that contained a contaminating subpopulationwith a different genotype, suggesting isolation of clonal cultures froma mixed population.

Both the manually and FACS-derived iPSC lines expressed common markersof pluripotency, including the surface marker Tra-1-60 and thetranscription factor Nanog, and generated compact coloniesmorphologically consistent with normal hESCs, FIGS. 19A-19B. Next, thecell lines were expanded for ten passages and the expression ofendogenous Nanog, Oct4, Sox2, cMyc, and Klf4, and silencing of the viraltransgenes Oct4, Sox2, Klf4, and cMyc were assayed. A probe-basedNanostring nCounter transcript quantification assay was used to assesspluripotency by detecting both activation of endogenous gene expressionFIG. 13A and silencing of retroviral transgenes FIG. 13B. These datawere further confirmed by qPCR FIGS. 19A-19D and revealed a similarpattern of endogenous gene expression in all iPSC lines compared toundifferentiated hESC controls FIG. 13A, FIGS. 19A-19D. However, two ofthe three manually derived clones (1018_2 and 1023_C) maintained muchhigher expression of the viral transgenes than the sorted clones FIG.3B. Additionally, the 1018_2 cultures expressed CD13, indicating thepresence of non-reprogrammed or partially transformed human fibroblastsin the manually picked lines FIG. 13B. These analyses suggest thatselection of single cells based on CD13NEGSSEA4POSTra-1-60POS expressioncan be used to select against partially reprogrammed or contaminatingcell types in reprogrammed cultures. The full data set for theseexperiments is provided in Table S7 (see Kahler et al, 2013,incorporated herein by reference).

Modified pluripotent scorecard assay was performed on manually and FACSderived clones to demonstrate (A) activation of endogenous geneexpression and (B) silencing of gene expression and presence ofunreprogrammed and transformed fibroblasts CD13POS in manually derivedclones. (C) Three sorted and three picked lines from patient 1023 wereused to compare the ability of both methods to generate independentclones. 10 μl of genomic DNA were cut overnight with BglII and submittedto Southern blotting. The HUES line HES2 was used as a positive controlfor endogenous KLF4/OCT4, and as a negative control for transgeneinsertions. Samples were first blotted for KLF4, then stripped andreblotted for OCT4. Picked clones 1023 C and E are consistent with beingthe same clone by both KLF4 and OCT4 blotting. * indicated the predictedendogenous KLF4/OCT4 bands, and ** indicated a consistent band found inall samples blotted with OCT4.

We next examined undirected EB formation to measure the in vitrodifferentiation potential of FACS and manually-derived iPSCs clones.Following differentiation for 5 weeks, EBs were collected and assayedfor markers of three embryonic germ layers endoderm, mesoderm, andectoderm by immunohistochemistry for α1-fetoprotein (AFP), smooth muscleactin (αSMA), and beta III tubulin (Tuj1), respectively. EBs derivedfrom FACS or manually picked clones expressed markers associated withformation of the three germ layers FIGS. 14A-14B. To further define thedifferentiation potential of the derived lines, RNA from the EBs werecollected after two weeks of differentiation and tested against a panelof lineage-specific nCounter probes Table S4 previously validated todetect expression of genes commonly found in the three germ layers [15]FIG. 14C. With the exception of the FACS-derived 0825 line, all linesexpressed comparable levels of the germ layer-associated genes,indicating they have similar potential to spontaneously differentiate invitro into any germ layer. The full data set for these experiments isprovided in Table S8 (see Kahler et al, 2013, incorporated herein byreference).

Embryoid bodies were derived from FACS (A) or manually derived clones(B) and stained for expression of alpha fetoprotein, smooth muscle actinand beta III tubulin (Tuj1) to demonstrate differentiation potential invitro potential. 10× Magnification (C) Differentiation potential of thederived lines for expression of germ layer genes present in the Lineagescorecard assay. (D) Teratomas from FACS (D) or manually derived (E)clones of 1023 fibroblast line indicating in vitro differentiationpotential by formation of three germ layers.

To measure the in vivo differentiation potential of the iPSCs,immunocompromised mice were injected with FACS or manually derivedclones from the 1023 fibroblast line. The resulting teratomas weresectioned and examined by H&E staining. This analysis showed thatteratomas generated from sorted FIG. 14D or manually derived FIG. 14Eclones formed all three germ layer tissues, including gut-likeepithelial tissues (endoderm), cartilage (mesoderm), and retinal pigmentepithelium (ectoderm). Together, these analyses validate the use ofCD13NEGSSEA4POSTra-1-60POS expression as a surface marker signaturecompatible with FACS that can be used to isolate a population of fullyreprogrammed iPSCs.

FACS Derivation Produces Independent Clones

Because iPSC lines can arise from rare apparently stochastic events atearly time points during reprogramming, it was important to establishthat FACS sorting could isolate multiple independent reprogrammingevents. To determine whether FACS derivation can produce unique celllines in a similar manner to manual picking, Southern blotting wasperformed on several clones derived by both methods. Klf4 and Oct4probes were used to identify the endogenous and virally integrated formsof the genes. Different banding patterns indicate differences in thechromosomal integration sites and, in some cases, varying numbers ofdetectable integration events. As shown in FIG. 13C, all three sortedclones from line 1023 have different integration sites for both Klf4 andOct4, demonstrating they are independent clones. In contrast, two of thethree picked clones from line 1023 have identical banding patterns forKlf4 and Oct4, suggesting they are the same clone. Of the iPSC linesgenerated from three different fibroblast lines, 8/9 FACS-derived lineswere independent clones, while 7/9 manually picked lines wereindependent (data not shown), suggesting equivalent ability to generateclonal cultures. Therefore, FACS sorting between 7-14 dpi using ofCD13NEGSSEA4POSTra-1-60POS can generate independent clonal culturesfollowing retroviral reprogramming.

FACS Derived Lines are Stable at Later Passages

To demonstrate the stability of FACS derived iPS clones at laterpassages, a foreskin fibroblast line (0819) was retrovirallyreprogrammed using both FACS and manual derivation methods. Threeindividual clones were chosen from each derivation method and expandedon MEFs to asses pluripotent surface marker expression by FCM at laterpassages. As shown in the first row of FIGS. 15A-15B, cultures of allclones (C1-C6) resulting from each derivation method possessedpopulations of cells positively expressing both SSEA4 and Tra-1-60 atvarying proportions indicating stability at between 12-14 passages.Although not shown in FIGS. 15A-15B, cultures of these clones werestable at earlier (p4-p11) and later (p20-p25) passages. Clones C3 andC6 were adapted to matrigel and mTser media (*C3 and *C6 in FIGS.15A-15B) following 11 passages on MEFS and expanded for 3-5 passages todemonstrate the stability of FACS derived iPS lines following changes insubstrate and media conditions. FCM analysis of Matrigel adapted iPSlines show stable surface marker expression with lessSSEA4POSTra-1-60NEG populations than the manually derived clone C6.Small populations of CD13POS expressing both Tra-1-60POS and Tra-1-60NEG(second row of FIGS. 15A-15B) were present in all cultures with theexception of the FACS derived C3 and *C3 clones indicating thevariability present in individual clones derived under DNA integratingreprogramming techniques. Similar results are observed within clonesderived using the non-integrating Sendai viral platform. These resultsdemonstrate that FACS derived iPS clones remain stable over multiplepassages and following adaptation to feeder free conditions.

Three individual clones were selected from foreskin (0819) fibroblastslines which previously underwent four factor retroviral reprogrammingand were derived by either FACS (A, C1-C3) or manual (B, C4-C6)techniques were analyzed by flow cytometry for pluripotent surfacemarker expression following expansion on murine embryonic fibroblastsfor 12-14 passages. Clones C3 and C6 were adapted to Matrigel and mTSERmedia around passage 11 and expanded for several passages prior tosurface marker analysis by flow cytometry to demonstrate stabilityfollowing changes in culture conditions. Events displayed in the 2Dscatter plots are “live” cells as defined by forward and side scatterproperties expressing indicated surface markers.

Utility for Multiple Reprogramming Methods

To further validate that the FACS surface marker panel can be used formultiple reprogramming methods, fibroblasts were reprogrammed usingnon-integrating Sendai viral constructs carrying the four Yamanakareprogramming factors and compared the FACS and manual derivationmethods to determine if there were differences between the integratingand non-integrating reprogramming systems. For these studies, an adultfibroblast line (131) was infected and subjected to either FACS sortingat 11 dpi or to manual derivation. At 11 dpi, the fraction ofSSEA4POSTRA-1-60POS cells that were also CD13POS was significantly lower(1-2%) than with retroviral reprogramming (37-54%, FIG. 12C), suggestingan accelerated rate of reprogramming. As before with the retrovirallines, several clones from each derivation technique were selected andexpanded for characterization following confirmation that the parentfibroblast line possessed a normal karyotype and DNA fingerprint, andwas free of contaminating cell lines (FIGS. 18A-18B).

As before, individual clones selected from both FACS and manualderivation techniques expressed the common markers of pluripotency, asrevealed by immunostaining FIGS. 16A-16D. In addition, the sorted (SRT)and picked (PCK) clones showed comparable levels of endogenous Oct4,Sox2, KLF4, MYC, and Nanog gene expression as early as passage 5, whichremained relatively constant to passage 10, FIG. 16E. Similarly, thoughgreatly reduced compared to control infected fibroblasts, Sendai virusgene expression was slightly above background at passage 5 in one sortedand one picked clone. However, this Sendai gene expression waseliminated by passage 10 in both cases, FIG. 16F. The full data set forthese experiments is provided in Table S9 (see Kahler et al, 2013,incorporated herein by reference).

Immunofluroescence microscopy of the 1001.131.01 line demonstratingexpression of common markers of pluripotency by FACS or Manually DerivedIPSC lines. Nuclear Transcription Factors shown in Green, SurfaceMarkers shown in Red, Nucleus stained with DAPI in Blue (A)Nanog/Tra-1-60 (B) Oct4/Tra-1-81 (C) Sox2/SSEA4 (D) Oct4/AlkalinePhosphatase. 10× Magnification (E) Expression of endogenous pluripotenttranscription factors (F) Silencing of viral transcription factors occurby passage 5. (G) Expression levels of transcription factors common tothe indicated germ layers from EB generated by the indicated IPSC lines.

The in vitro differentiation potential of FACS and manually derived iPSCclones reprogrammed by the Sendai virus protocol was evaluated by EBformation and a lineage specific nCounter assay as above. Similar trendsin gene expression were observed for clones derived under both methods,FIG. 16G. Both sorted and picked lines expressed levels of theectodermal marker FGFR2 comparable to that of the control HUES42 linebut higher levels of the mesodermal marker HHEX. Most clones showedlevels of endodermal gene expression comparable to the HUES42 line withthe exception of a manually derived clone that expressed higher levelsof GATA6 than the remaining picked lines or FACS-derived lines. The fulldata set for these experiments is provided in Table S10 (see Kahler etal, 2013, incorporated herein by reference). Collectively, these datademonstrate that using FACS to purify the CD13NEGSSEA4POSTra-1-60POScells from either retroviral or Sendai viral 4-factor reprogrammingprotocols consistently produces high quality iPSC lines.

DISCUSSION

Clinical application of iPSC technology will require standardized andreproducible methods for each step of derivation and differentiationinto relevant cell types. The majority of manually derived iPSC lineswere found to contain CD13POS cells even after prolonged culturesuggesting that these lines were either not fully reprogrammed or thatCD13POS cells were carried over during passage. While both manualpicking and FACS sorting methods [10] have been used to isolatereprogrammed pluripotent cells, inclusion of a negative selection markersuch as CD13 has significant advantages in improving the purity ofreprogrammed cultures. This fact was previously demonstrated [13]. Here,findings have validated a surface marker profile that enables selectionof early reprogrammed iPSCs following reprogramming with eitherDNA-integrating or non-integrating viruses by FACS. Employing thisstrategy as early as 7 dpi isolates a highly purified startingpopulation of fully functional CD13NEGSSEA4POSTra-1-60POS cells that aredepleted of contaminating non-transduced and transformed fibroblasts.228 individual iPSC lines have been successfully generated andcharacterized in 2 years from 76 fibroblast lines obtained from freshbiopsies, frozen stocks, and cell line repositories harvested fromhealthy and individuals possessing various forms of diabetes,neurodegenerative, cardiac and autoimmune diseases. Table 8, below.

TABLE 8 Summary of FACS Derived hIPSC Lines (S6) Total Model* Cell LinesDerivations Retro Sendai Alzheimer 11 20 19 1 Parkinsons 4 8 2 6 FTD 2 20 2 GAN 5 14 14 0 Cardiac_LMNA 3 7 6 1 Cardiac_LongQT 6 14 14 0 MODY 1134 24 10 T1D 3 24 17 7 T2D 1 8 8 0 MS_RR 1 2 0 2 MS_SP 1 1 0 1 Control28 94 51 43 Totals 76 228 155 73 *MS_RR Multiple Sclerosis RelapsingRemitting, MS_SP Multiple Sclerosis Secondary Progressive, FTD FrontalTemporal Dementia, GAN Giant Axonal Neuropathy, LMNA Lamin A/C, MODYMature Onset Diabetes of the Young

Moreover, FACS is routinely used for maintenance of established celllines to remove differentiated cells and to dispense graded numbers ofhighly purified CD13NEGSSEA4POSTra-1-60POS populations cells for use inhigh-throughput derivation and screening assays which include directeddifferentiation and automated drug screening and phenotypingexperiments. This is an important property because the results of theseassays could be unequivocally attributed to a defined population ofreprogrammed cells rather than to a heterogeneous mixture of cells.Taken together, these results suggest that isolation of theCD13NEGSSEA4POSTra-1-60POS population following reprogramming, includingintegrating or non-integrating viral technologies, allows for the rapidisolation of high quality iPSC lines. Negative selection against CD13POScells significantly reduces the appearance of transformed cells in ipsccultures and suggests that negative selection for a marker present onthe starting somatic cells can be used to exclude non-reprogrammed ortransformed cells from the cultures. Future studies will be needed todetermine if this strategy applies to derivation from other somatic celltypes or reprogramming methods.

REFERENCES FOR EXAMPLE 3

-   1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem    cells from mouse embryonic and adult fibroblast cultures by defined    factors. Cell 126: 663-676. doi: 10.1016/j.cell.2006.07.024.-   2. Kiskinis E, Eggan K (2010) Progress toward the clinical    application of patient-specific pluripotent stem cells. J Clin    Invest 120: 51-59. doi: 10.1172/JCI40553.-   3. Lee G, Papapetrou E P, Kim H, Chambers S M, Tomishima M J, et    al. (2009) Modelling pathogenesis and treatment of familial    dysautonomia using patient-specific iPSCs. Nature 461: 402-406. doi:    10.1038/nature08320.-   4. Maehr R, Chen S, Snitow M, Ludwig T, Yagasaki L, et al. (2009)    Generation of pluripotent stem cells from patients with type 1    diabetes. Proc Natl Acad Sci USA 106: 15768-15773. doi:    10.1073/pnas.0906894106.-   5. Zhu S, Li W, Zhou H, Wei W, Ambasudhan R, et al. (2010)    Reprogramming of Human Primary Somatic Cells by OCT4 and Chemical    Compounds. Cell stem cell 7: 651-655. doi:    10.1016/j.stem.2010.11.015.-   6. Inoue H, Yamanaka S (2011) The Use of Induced Pluripotent Stem    Cells in Drug Development. Clin Pharmacol Ther 89(5): 655-661.-   7. Smith Z D, Nachman I, Regev A, Meissner A (2010) Dynamic    single-cell imaging of direct reprogramming reveals an early    specifying event. Nat Biotechnol 28: 521-526. doi: 10.1038/nbt.1632.-   8. Sorrell J M, Baber M A, Brinon L, Carrino D A, Seavolt M, et    al. (2003) Production of a monoclonal antibody, DF-5, that    identifies cells at the epithelial-mesenchymal interface in normal    human skin. APN/CD13 is an epithelial-mesenchymal marker in skin.    Exp Dermatol 12: 315-323. doi: 10.1034/j.1600-0625.2003.120312.x.-   9. Chan E M, Ratanasirintrawoot S, Park I H, Manos P D, Loh Y H, et    al. (2009) Live cell imaging distinguishes bona fide human iPS cells    from partially reprogrammed cells. Nat Biotechnol 27: 1033-1037.    doi: 10.1038/nbt.1580.-   10. Valamehr B, Abujarour R, Robinson M, Le T, Robbins D, et    al. (2012) A novel platform to enable the high-throughput derivation    and characterization of feeder-free human iPSCs. Sci Rep 2: 213.    doi: 10.1038/srep00213.-   11. Noggle S, Fung H L, Gore A, Martinez H, Satriani K C, et    al. (2011) Human oocytes reprogram somatic cells to a pluripotent    state. Nature 478: 70-75. doi: 10.1038/nature10397.-   12. Boulting G L, Kiskinis E, Croft G F, Amoroso M W, Oakley D H, et    al. (2011) A functionally characterized test set of human induced    pluripotent stem cells. Nat Biotechnol 29: 279-286. doi:    10.1038/nbt.1783.-   13. Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, et al. (2009) A    chemical platform for improved induction of human iPSCs. Nat Methods    6: 805-808. doi: 10.1038/nmeth.1393.-   14. Pruszak J, Sonntag K C, Aung M H, Sanchez-Pernaute R, Isacson    O (2007) Markers and methods for cell sorting of human embryonic    stem cell-derived neural cell populations. Stem Cells 25: 2257-2268.    doi: 10.1634/stemcells.2006-0744.-   15. Bock C, Kiskinis E, Verstappen G, Gu H, Boulting G, et    al. (2011) Reference Maps of human ES and iPS cell variation enable    high-throughput characterization of pluripotent cell lines. Cell    144: 439-452. doi: 10.1016/j.cell.2010.12.032.-   16. Sundberg M, Jansson L, Ketolainen J, Pihlajamaki H, Suuronen R,    et al. (2009) CD marker expression profiles of human embryonic stem    cells and their neural derivatives, determined using flow-cytometric    analysis, reveal a novel CD marker for exclusion of pluripotent stem    cells. Stem Cell Res 2: 113-124. doi: 10.1016/j.scr.2008.08.001.-   17. Ng V Y, Ang S N, Chan J X, Choo A B (2010) Characterization of    epithelial cell adhesion molecule as a surface marker on    undifferentiated human embryonic stem cells. Stem Cells 28: 29-35.    doi: 10.1002/stem.221.-   18. Lu T Y, Lu R M, Liao M Y, Yu J, Chung C H, et al. (2010)    Epithelial cell adhesion molecule regulation is associated with the    maintenance of the undifferentiated phenotype of human embryonic    stem cells. J Biol Chem 285: 8719-8732. doi:    10.1074/jbc.M109.077081.-   19. Henderson J K, Draper J S, Baillie H S, Fishel S, Thomson J A,    et al. (2002) Preimplantation human embryos and embryonic stem cells    show comparable expression of stage-specific embryonic antigens.    Stem Cells 20: 329-337. doi: 10.1634/stemcells.20-4-329.

Example 4 Generation of Large Genetically Diverse Cell Panels

A tissue procurement process has been established through which over 500genetically diverse skin samples have been collected representingapproximately 79% of the ethnic diversity needed to model the U.S.population (based on U.S. census data). 500 fibroblast lines have beengenerated from these samples and 300 iPSC lines using an automatedsystem as described herein, with work continuing to generate more linesand to expand the sample set further. The cell lines produced areprovided in 96-well microtiter plates with each well containing cellsderived from a different individual.

Somatic cells are isolated, expanded, and analyzed for copy numbervariations (CNVs) to determine karyotype and a genomic identifier.High-content imaging is used to collect data cellular growth rates, cellcounts, and morphology that guide reprogramming and differentiation.Non-genomic integrating reprogramming is accomplished using transductionof five individual mRNAs expressing Oct4, Sox2, Klf4, Lin28, and cMyc(see, for example, Warren et al., 2010). The process is performed in achemically-defined, xeno-free, media to minimize potential sources ofvariability in resulting cell populations. Clonal selection is achievedusing a multi-stage process including a magnetic bead based enrichmentscheme where non-reprogrammed cells are depleted from the cultures ofiPSCs by negative selection of CD13 and high-content imaging of surfacemarker Tra-1-60. The resulting iPSCs are transferred to a 96-well platewhere colony growth is monitored using the automated high-contentimager. For further characterization of the iPSCs, transcriptionalanalysis is performed by direct mRNA measurements of a set of 100 geneprobes covering pluripotency, all three germ layers, sex, andhousekeeping pathways.

This analysis is performed on the iPSCs as well as embryoid bodies (EBs)generated from them.

Example 5 A Fully Automated, High Throughput Platform for IPS CellDerivation and Characterization

Induced pluripotent stem cells (iPSCs) have become an essential tool formodeling how causal genetic variants impact cellular function in diseaseand are an emerging source of tissue for transplantation medicine.Unfortunately, the preparation of somatic cells, their reprogramming andthe subsequent verification of iPSC pluripotency are laborious, manualprocesses that limit the scale and level of reproducibility of thistechnology. The present Example describes a robotic platform for iPSCreprogramming that enables high-throughput conversion of skin biopsiesinto iPSC lines with minimal human intervention. Importantly, iPSC linesmanufactured by this automated system exhibited significantly lessvariation than those produced manually. This robotic platform thusenables the application of iPSCs to population-scale biomedical problemsincluding the study of complex genetic disease and the development ofpersonalized medicines.

INTRODUCTION

The reprogramming of somatic cells into induced pluripotent stem cells(iPSCs), coupled with the development of methods for directing stem celldifferentiation into relevant cell types, offers an unprecedentedopportunity to study the cellular phenotypes that underlie disease(Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Rubin, 2008;Colman and Dreesen, 2009; Nishikawa, Goldstein and Nierras, 2008; Daley,2010). Study of these emerging “stem cell disease models” has led to newmechanistic insights into a wide variety of conditions ranging fromneurological disorders to cardiovascular and infectious disease(Robinton and Daley, 2012).

One limitation of the application of these methods is the substantiallevel of variation between different stem cell lines that arises duringthe reprogramming progress, affecting both functional properties of thelines and their performance in disease-related studies. As a result,most successful reports have relied on evaluation of several cell linesderived from individuals harboring genetic variants of high penetrance.If stem cell technologies are to be applied to the study of commongenetic variants that confer only modest effects but have been shown tobe important contributors to conditions such as schizophrenia, andmetabolic disease (Morris et al., 2012; Ripke et al., 2013), it will beessential to minimize variation arising during reprogramming, subsequentstem cell expansion and differentiation. Reduction in this source ofvariation would increase resolution of the phenotypic effectscontributed by the genotype of a particular individual.

Presently, it remains unclear what fraction of or the extent to whichvariation between iPS cell lines arises from technical aspects of theprocedure, rather than from uncontrollable, stochastic epigenetic eventsthat occur during reprogramming. Possible contributors to functionalvariation include genetic background and tissue source, {reviewed in(Cahan and Daley, 2013; Nestor and Noggle, 2013); reprogramming factorstoichiometry during delivery (Carey et al., 2011); and stress duringculture (Liang and Zhang, 2013). Additionally, a growing number ofmethods are used to deliver reprogramming factors and various cellculture media and substrates have been used for downstream expansion(Chen et al., 2014). This lack of standardization likely introducesadditional variability among cell lines established by differentlaboratories (Newman and Cooper, 2010). As automation has improvedprecision and scalability in many areas of biomedical research (Meldrum,2000; Meldrum, 2000; Lander et al., 2001), developing a fully automatedplatform for iPS cell derivation, expansion and differentiation wouldallow identification of and restriction of the factors contributing tovariability in stem cell line behavior.

The present Example describes the development of three integratedrobotic systems that automate the entire process of deriving iPSC lines.This integrated reprogramming platform was used to systematicallyexplore several variables that have been reported to be importantsources of variation in the reprogramming process. Importantly,automation alone was found to eliminate a significant portion of thevariation in differentiation propensity exhibited between iPS celllines.

Results Automated Fibroblast Production

As a first step in developing an integrated and automated process forderiving iPSCs (FIG. 20A), a robotic system has been established forisolating skin fibroblasts from volunteer biopsies and tissue samples(FIG. 20A, Stage 1). This system processes tissue under quarantineconditions prior to mycoplasma testing, allowing for the expansion andfreezing of fibroblasts before being transferred into a cleaner cultureenvironment. The system is housed in a BSL II biosafety hood (FIG. 20B)and integrates a robotic liquid handler with an automated centrifuge,microscope and incubator. The biopsy processing system is complementedby a second, independent liquid handling robot connected to aluminescence plate reader, used to conduct mycoplasma detection assays.

For fibroblast derivation, biopsies and tissue samples were manuallydissected and placed into individual plates filled with a low serumculture media selected to eliminate batch to batch variation in serum.In addition, low serum medium is more compatible with down-streamreprogramming using modified RNAs (Warren et al., 2010). All furthermaintenance, including passaging and feeding, was performed viaautomation. Every five days biopsy outgrowth was measured throughautomated image acquisition and quantification (FIGS. 20C and 20D).Media exchanges and media collection for mycoplasma testing were alsohandled in an automated fashion. Multi-well plates of media collectedfor mycoplasma testing were then placed onto the second platform forautomated testing (FIG. 27A). Following completion of mycoplasmatesting, fibroblast outgrowths were enzymatically passaged by the systeminto new plates for further expansion.

After one passage, in order to minimize population doublings andincrease reprogramming potential (Utikal et al., 2009), fibroblasts werefrozen into multiple 2D barcoded cryovials and banked to produce lowpassage frozen stocks. The automated imaging system first identifiedwells with outgrowth (FIG. 20Di) and area of the culture plate occupiedto calculate a confluence value (FIG. 20Dii). In some experiments, DNAstaining and algorithms were used for identifying and counting nuclei toextrapolate cell counts from confluence values using standard curves(FIGS. 20Diii and 20Div). This allowed non-invasive calculation ofgrowth rates of the fibroblasts. As early experiments suggested thatfewer than 100,000 cells were needed for reprogramming, yields offibroblasts from outgrowths at this early stage of growth weresufficient for the subsequent reprogramming steps (778,216 cells perpassaged well of a 6-well dish s.d.=88,813, n=60). This allowed thecells to be frozen at the second passage. One vial was typicallyreserved for iPS generation through short-term storage within anautomated −80° C. sample access manager, with the remainder banked inliquid nitrogen. An average of 363,000 fibroblasts were produced persample (s.d. of 305,000, n=168) with minimal expansion (passaging of thebiopsy outgrowth results in p1 cultures). The average total number ofcells frozen into a single cryotube at the time of freezing was 121,437cells which upon thawing had an average viability of 84%±1.43%(mean±SEM, n=167).

During development of the automated system, a total of 640 skin tissuesamples were processed with 89.4% producing fibroblasts that weresuccessfully frozen (n=572). Failures that arose were primarily due toeither bacterial or fungal contamination (4.7%, n=30) attributable tosample handling before entering the system, and were minimallyassociated with equipment or manual process failures (3.2%, n=21). Only2.7% (n=17) did not show outgrowth, which could be attributed to thesmall size of the biopsy, lacking an overt dermal layer. Using aNanostring nCounter karyotyping assay that can detect aneuploidy as wellas large chromosomal gains/losses (FIG. 27B), 20 randomly selected,representative fibroblast samples were tested. The majority of samples(19/20, 95%) showed a normal diploid karyotype (46XX or 46XY). Thus, theautomated process can successfully derive, expand and cryopreserve highquality, low passage fibroblasts which can be banked for furtherprocessing or future use.

To monitor fibroblast growth rates and cell culture densities of theresulting plates, the automated imaging system was used (FIG. 20Dii).Various approaches were tested for monitoring fibroblast growth andfound that confluence measurements (percent of well covered by cells)were sufficient to allow reliable tracking of growth rates. Frequentmeasurements allowed for doubling time calculations to be performedduring log phase growth. Under these low-serum conditions, averagefibroblast doubling time was 66 hours (s.d.=30.2 h, n=298). (FIG. 20E).This modest doubling time is not believed to be a product of growth onthe automated system per se as when a subset of these samples wasrobotically fed with serum-containing media the doubling time wasdecreased to 45.8 hr (s.d.=16.5 h, n=33). While the mean doubling ratesdid not significantly differ from their growth rates prior to freezing(p=0.69), the variation decreased by almost half upon recovery in serum(% CV=61% before vs. 36% after thaw) (FIG. 20G). As doubling rate didnot correlate with age (FIG. 20F), this suggests it should be possibleto group samples with similar growth characteristics soon after initialoutgrowth to establish cultures for downstream automated reprogrammingas described below.

Automated iPS Reprogramming

To enable automated reprogramming, an integrated robotic platform wasconstructed with the capacity to thaw fibroblast samples, deliverreprogramming factors via Sendai virus transduction (Fusaki et al.,2009) or modified mRNA transfection (Warren et al., 2010), performmagnetic selection of reprogrammed cells, and finally image cultures toidentify nacent stem cell colonies after surface marker staining (FIG.20A, Stage 2, and FIG. 21A).

First, a viral infection method using Sendai virus (Fusaki et al., 2009)was adapted to the automated liquid handling system. Automated deliveryof Sendai virus to 50,000 fibroblasts at a moiety of infection of 4resulted in 2 to 10 TRA-1-60 positive colonies per well under feederfree conditions. Similar efficiencies were obtained by manual deliveryunder these conditions. Several lines were derived by manual picking ofTRA-1-60 positive colonies after automated infection and appearance ofcolonies (FIG. 28B) before continuing the culture of the picked colonieson the automated system. Established cultures from these coloniesexpressed common markers of pluripotency, including Nanog, Oct4, SSEA4and TRA-1-60 (FIG. 28C). Consistent with previous results (Kahler etal., 2013), automated Sendai infections induce TRA-1-60 positive cellsthat retain surface expression of a fibroblast marker with an average of58.2% (n=12) of TRA-1-60+/SSEA4+ were also CD13+(FIG. 28D). Further,after differentiation in EB assays (see below), significant clonalvariability was found in gene expression that appeared to coincide withresidual Sendai transgene expression remaining in the lines betweenpassages 5 and 10 (FIG. 28E). Between passages 11 and 12, the Sendailines were incubated at 38.5° C. to encourage Sendai inactivation(specifically C-Myc) at which point a decrease in Sendai virusexpression was observed. However, after a further 2-3 passages, thelines were reanalyzed and Sendai transgene expression had againincreased. Additionally, variability in toxicity was noted amongdifferent lots and batches of virus. Together, these results suggestedthat, while it is possible to use Sendai virus to reprogram fibroblastsby automated methods, it was unlikely to emerge as a preferred method.

Next, transfection methods were adapted to deliver modified mRNA (Warrenet al., 2010) to reprogram fibroblasts by automation. After automatedpassaging, fibroblasts were treated via automated media exchange with B18R to block interferon response. The liquid handling system was alsoused to deliver transfection mix containing miRNA (Miyoshi et al., 2011)followed by 10 daily transfections of base modified mRNA encoding Oct4,Klf4, Sox2, c-Myc, Lin-28, and nuclear GFP (nGFP) (FIG. 21B). The miRNAwas also included together with the fourth mRNA transfection. Humanfibroblast conditioned media was replaced daily by automated mediaexchanges before each transfection. Six days after final transfectionthe cultures were transitioned to a feeder-free hES cell culture mediumusing automated partial media exchanges. This integrated automatedprocess was tested by performing 20 consecutive experimentalreprogramming runs of 24 fibroblast samples in duplicate and undervarying conditions, for a total of 960 independent reprogrammingexperiments (FIG. 21A).

In addition to adult donor fibroblasts, BJ foreskin fibroblasts wereincluded in each run to control for run to run variability. Followingthis protocol, nascent iPSC colonies were consistently observed between16 and 22 days of culture (FIGS. 21C-21D). Established culturesdemonstrated pluripotent hESC-like morphology and expressed commonmarkers of pluripotency, including Nanog, Oct4, Sox2, SSEA4 and TRA-1-81(FIG. 28A). Over all runs, an average of 7 TRA-1-60+ colonies wereinduced per well (n=299, range 1-40 colonies). These cultures containeda high proportion of TRA-1-60+/SSEA4+ cells and an average of 9.7%(n=38) of cells retained CD13 (FIG. 21E). Reprogramming efficiency wasbetween 0.001% and 0.16% for successful attempts per plated somaticcell, consistent with previous results obtained under feeder freeconditions (Warren et al., 2012), and was slightly higher for control BJfibroblasts (0.43%) than for adult fibroblast cultures (0.014%).

The next analysis investigated the factors that correlated withreprogramming success rates. The number of colonies produced per wellwas determined after mRNA mediated reprogramming production runs andseveral parameters were compared during fibroblast recovery andreprogramming. The number of colonies was determined by automated imagebased colony identification and counting after TRA-1-60 live cellsurface staining of the cultures (FIG. 21D). Although fibroblasts fromolder donors were successfully reprogrammed, increasing donor agenegatively influenced the number of colonies produced. Additionally,slower proliferation rates during reprogramming as well as the presenceof serum during fibroblast recovery and passaging all had a negativeimpact on the number of colonies produced, when controlling for othervariables included in the analysis (FIG. 21F-I). This is consistent witha transition from serum containing media to serum-free media reducingproliferation rates important for reprogramming success. Together, theseresults define conditions that allow for the derivation of iPSCs usingthis automated process.

Automated iPS Purification

FACS methods have previously been described for enriching reprogrammediPSCs using a standard panel of cell surface markers (Example 3 above,and Kahler et al., 2013). In particular, incompletely reprogrammed cellsoften retain surface expression of fibroblast specific markers. Toachieve a greater throughput, reprogrammed iPS cells were enriched usingautomated magnetic bead based negative selection to remove incompletelyreprogrammed cells (FIG. 22A). Through the integration of a MultiMACSmulti-well magnetic bead separation device into the liquid handlingsystem, negative selection was used to, in parallel process 24 lines ata time (FIG. 22B). To test the system, spike in experiments wereperformed with an iPS cell-to-fibroblast ratio of between 1:20 and 1:100that demonstrated that a 6-fold fibroblast depletion and an average47-fold iPS cell enrichment could be achieved (FIG. 29A). In practice,the magnetic bead selection process resulted in an average of ˜30%CD13-/SSEA4+/TRA-1-60+ cells following completion of the automated mRNAreprogramming protocol (FIG. 29B).

The population flow-through fraction of iPS cells by MACS was thenplated into 96-well plates. The flow-through fraction was plated on anextracellular matrix in serum and feeder free conditions in a threepoint, two-fold serial dilution in 12 wells per initial reprogrammedwell to recover colonies (FIG. 22D). Starting as single cells at thefirst day after sorting, TRA-1-60+ colonies formed within 7-10 days(FIG. 22C) and had a typical ES cell morphology (cells with largenuclei, small amount of cytoplasm, with compact monolayer colonymorphology). Doubling times, calculated from daily confluencemeasurements using the automated imaging system, ranged from 24-48 hrsfor most lines. Sorted cells were checked daily using the Celigoautomated microscope. Doubling times for most lines ranged from 24-48hrs (FIG. 22G). Additionally, it was retrospectively established thatclonal iPSC lines arose from expansion of single cells plated in singlewells (FIGS. 22E and 29C). After wells with uniform confluence valueswere identified, three candidate wells per sample were selected andconsolidated 1 well to 1 well onto 96-well plates by the robotic liquidhandling methods for expansion and further quality control assays. Afterthis passaging step, flow cytometry analysis further demonstrated that˜80% of the cells were SSEA4+/TRA-1-60+(FIG. 22F). These passaged iPSCsreestablished typical human ES cell like colony morphology and expressedthe pluripotent marker NANOG (FIG. 30B). To further ensure the negativeselection had eliminated non-reprogrammed or partially reprogrammedcells, the sorted cultures were further screened by gene expressionassays using a panel of pluripotency (NANOG, POU5F1, LIN28, ZFP42, SOX2)and differentiation (ANPEP, NR2F2, AFP, SOX17) marker genes. Using ananalysis strategy similar to one previously described (Bock et al.,2011), performance of candidate iPSC lines were compared to anestablished reference panel of hESC lines (FIGS. 23A, 23B). A set ofnewly reprogrammed and sorted iPSC lines were tested using this assayand 9 of 11 tested lines had scores consistent with the hESC referencepanel. Two of the lines tested showed pluripotency scores consistentwith reference standards, but showed elevated differentiation scores(FIG. 30A). This could be attributed to overgrowth of iPSCs resulting inspontaneous differentiation in these cultures (FIG. 30C). Together,these data suggest that iPSC lines purified by automated processesdemonstrated characteristics of reprogrammed pluripotent cells withintwo passages after reprogramming. Thus, candidate iPSC lines in 96-wellformat can be recovered by automated single well passaging methods toconsolidate and array multiple iPSC lines in parallel for expansion andfurther analysis.

Automated Culture of Multiple iPSC Lines in Parallel

The primary aim for developing the automated process was to allowparallel derivation and culture of multiple iPS cell lines in parallel.The knowledge of growth rates and cell density at the time ofconsolidation combined with the ability to bin and batch cell linesand/or adjust plating characteristics at various steps was hypothesizedto further enable parallel culture of cell lines with diverse growthcharacteristics at early passages (FIG. 22G). To enable this, automatedand informatic processes were developed for cryopreservation of nascentiPSCs with similar growth characteristics (FIG. 20A Stage 3, and FIGS.24A and 31A). After freezing in cryotubes followed by subsequent thaw,iPSCs recovered to show a normal morphology (FIG. 24B). Correlationsbetween pre-freeze and post-thaw recovery confluencies were highest oneday after the thaw (Pearson's r>0.91) and they decreased as the wellsapproached full confluence (Pearson's r>0.71 on day 3, >0.41 on day 6),due to overgrowth of higher confluence wells (FIG. 24C). The percentageof SSEA-4/Tra-1-60 double positive iPSCs remained consistent before andafter the thaw (FIG. 24E), with subsequent passaging showing nosignificant differences in the percentages of the SSEA-4+/Tra1-60+double positive population (FIGS. 24D and 31D). Enzymaticallyrobotically passaged cultures formed morphologically ideal colonies withwell defined edges and expressed pluripotency markers POU5F1, Oct-4,Sox2, Nanog, SSEA-4, Tra1-60 and Tra 1-81 (FIG. 31C). The culturesgrowing in 96-well plates could be successfully maintained over 5-7 daysgrowth and before requiring passaging (FIG. 24F). Following expansion ofa single 96-well plate into three plates, between-plate variationremained low (FIG. 31B). Additionally, the wells did not loseSSEA-4+/Tra1-60+ pluripotency marker expression (FIG. 31D). Passagingrations between 1:1 and 1:15 have been successfully performed on theautomated system. Together, these data indicate that after freezing, thegrouping and thawing of newly derived iPSC lines, samples cansuccessfully occur based on prior growth characteristics. Subsequentexpansion of these lines does not alter either the growth orpluripotency characteristics allowing the automation of parallel culturein a multiwall format.

Analysis of genomic DNA was performed to track cell line identity (FIGS.31F-31H) and to ensure that cell lines remained karyotypically normal(FIG. 31E). iPS lines were karyotyped using the Nanostring nCounterkaryotyping assay that can detect aneuploidy as well as largechromosomal gains/losses commonly found during manual pluripotent stemcell culture. Whilst previous reports have stated that chromosomalabnormalities arise in approximately 20% of iPSCs (Mayshar et al.,2010), the majority of the presently described iPSCs (89%, n=38) showeda normal diploid karyotype (46, XX or 46, XY). Of the 4 abnormal linesdetected, one had a chromosomal gain in the long arm of chromosome 22(probes spanned 22q11.21-22q13.32). The remaining 3 abnormal lines allshared a common starting fibroblast population and shared a copy numbergain in chromosome 17 (probes spanned 17q21.32-17q25.3), suggestingheterogeneity of the original fibroblast population that was below thethreshold of detection. Together, these results suggest that it ispossible to maintain iPSCs during subsequent automated passaging.

Automated Analysis of Differentiation Propensity

To assess the differentiation potential of robotically derived iPSCs, arobotic platform was developed to generate EBs and the ability of iPSCsto spontaneously differentiate into the three germ layers was measured.Several automation compatible methods for reproducibly forming EBs weretested followed by an automated process to harvest and lyse EBs beforeusing a Nanostring gene expression assay to quantitatively assess germlayer differentiation as previously described (Bock et al, 2011). Theprevious assay was modified to include the 83 germ layer lineage genesdivided into ectoderm (EC), mesoderm (ME), and endoderm (EN) gene setsas well as probes for transgene silencing and Sendai vector elimination,sex (SRY and XIST), pluripotency (Oct4, POU5F1, Sox2, Nanog, ZFP42), andhousekeeping gene expression (ACTB, POLR2A, ALAS1). In initial pilotstudies, hierarchical clustering of EB gene expression suggested thatthat samples group together according to the type of method used for theEB formation assay. EBs formed by hanging drop methods clusteredseparately from those produced by plating in V-bottom plates. Expressionof these gene sets was measured relative to those from a control panelof EBs made from hESC lines analyzed in parallel under identical cultureconditions (FIGS. 32A-32B). Pluripotency of the hESC lines used forreference standards was verified by marker staining for POU5F1, Oct4,TRA-1-80, Nanog, SSEA4, SOX2, TRA-1-60, and Alkaline Phosphatase (FIG.32A). All lines tested exhibited scorecard differentiation propensitiesconsistent with the ability to differentiate into the three germ layers(FIG. 32B). Germ layer lineage marker gene sets were differentiallyexpressed between the different methods of forming EBs (FIG. 25A), withhanging drop methods generating gene expression biased towards endodermand V-bottom plates demonstrated a more uniform differentiationpotential. Together, this suggests that different methods of generatingEBs may confound comparisons among lines and highlights the need formethod standardization.

EB formation was optimized using EGFP marked iPSC lines, to seed cellsfrom a single well of a 96-well plate into six wells of 96 well V-bottomplates (FIGS. 25B-25C). Before EBs were collected, plates were imagedusing Celigo to monitor EBs formation (FIG. 25B). These automatedmethods allowed the iPSCs to aggregate and form embryoid bodies at thebottom of the well (FIG. 25C). While the differentiation propensityscorecard values for all samples showed excellent correlation with thosepreviously published (Bock et al., 2011), a slight decrease was noted incorrelation with previously published data from identical referencelines grown under different culture conditions (FIGS. 25D-25F),suggesting that the assay is sensitive to differentiation cultureconditions. Therefore comparisons were made using reference datagenerated from hESCs adapted to the culture conditions used to deriveiPSCs on the automated system.

Reduced Variation in Robotically Derived iPSCs

Next, the propensity of robotically derived iPSCs to differentiate bythe automated EB assay was measured and lineage scorecard analysis wascarried out described above was used (Bock et al., 2011). Hierarchicalclustering of gene expression shows overall consistency of the IPSCsgenerated by automation. However, the lineage scorecard differentiationassay suggested that it might be possible to distinguish amongvariations in methods used to derive the iPSC lines independent ofgenotype. It was found that iPSCs derived through complete automation ofthe reprogramming process showed reduced variation (as measured bycomparing distributions of standard deviation of gene expression) whencompared to lines reprogrammed by manual processes (p value=7.08×10−12,Wilcoxen signed rank test) (FIG. 26). This was true in comparisonswithin a single genotype (BJs, p values=7.85×10−9) and as well as forthe patient lines (donor, p values=9.28×10-11). Interestingly, iPSCsinitially reporgrammed robotically before having colonies manuallypicked and then returned to the automated system for expansion showedelevated expression similar to that found in existing manually derivediPSC lines (p value=0.023. Thus this finding suggests that manual cloneselection may be an important source of variation. Additionally, linesderived by Sendai infection and manual colony picking showed similarvariation to lines initially reprogrammed using automation but completedthrough manual colony picking (p value=0.40). This suggests that linesproduced by the completely automated process show reduced variation atearly time-points after derivation compared to lines derived by currentmanual procedures.

DISCUSSION

This Example describes an embodiment of a fully automated platform forreprogramming easily obtainable skin cells into iPSCs. The describedplatform achieves reproducibility and population scale iPSC derivationand differentiation, using an automated approach based on recentadvances in reprogramming and characterization methods (Bock et al.,2011; Kahler et al, 2013; Warren et al, 2010). A fully-robotic processhas been established for the generation of fibroblast banks, iPSCreprogramming, as well as automated assays for assessing differentiationpotential. The creation of this platform allowed assessment of thesources of functional variability between iPS cells. Importantly, sue tothe use of robotics, this has been achieved with both the precision andscale that could previously not have been considered.

Most notably, manual selection of newly reprogrammed iPS cell colonieswas in itself found to be a substantial contributor to cell line to cellline variation. Through automation of the reprogramming process, morethan a third of the variability that existed between manually selectedlines was eliminated. This finding demonstrates that at very least, asubstantial portion of this variation had purely technical origins.

The large scale of the described experiments also allowed investigationof the previously raised question of whether the origin of the somaticcells and and/or their genetic background, influenced stem cell linebehavior. The production of many cell lines from both a single somaticpopulation (BJ fibroblasts) and from multiple distinct individualsallowed investigation of this issue. The results presented in thisExample clearly show that the level of variability between cell linesmade from many donors was not different from that found with lines froma single donor. Previous studies suggest that genetic factors could be acontributing factor to functional variance between iPS cell lines(Kajiwara et al., 2012). However, this data suggests that if thesefactors do contribute, the do so modestly in comparison to the technicalvariation that can be resolved through automation.

Although it has been cited as a potential inhibitor of reprogramming inthe past, advancing age of the subject being reprogrammed was not asignificant modifier of reprogramming efficiency. Instead, both thegrowth rate and confluence of cell cultures at the time of reprogrammingwere found to be the primary drivers of whether the automated approachsucceeded in producing iPS cells in each individual case. This findingseems consistent with the observation that genetic factors which slowproliferation of cells inhibit reprogramming (Hanna et al, 2009).

In addition to this, the reprogramming method used for producing iPSCshad a substantial effect on cell line properties. Although aspects ofiPSC production using both mRNA delivery and Sendai virus infectioncould be automated, incomplete eviction of Sendai virus led to asubstantial change in the signature of gene expression in pluripotentcells. For this reason, plus previously discussed issues with lot to lotvariation and availability, a modified mRNA reprogramming method as astandard protocol. However, the flexibility of the system allows for thefuture adoption of other reprogramming methods as these becomeavailable.

As described here, the integrated system has a capacity to produceapproximately 384 iPSC lines per month. Running a second personnel shiftusing the current system could allow capacity to double, resulting inthe ability to produce 768 iPSC lines per month. The advantage to thisapproach of automating production over a manual process, however, isthat capacity can be scaled with additional systems with only a minimalincrease in personnel time. The timeline for producing seed stocks ofcharacterized iPSC lines from fibroblasts is approximately 12 weeks.Although the current automated system may not yet be ideal for clinicalgrade iPSC, evidence that the process can be automated suggests that asimilar system tailored to the clinical grade production is nowfeasible. Together this increased scale and accelerated timeline shouldenable many large-scale projects utilizing iPSCs (McKernan and Watt,2013).

In the future, the increased throughput of reprogramming and reducedvariability between the resulting lines should open a number of newavenues for investigation. First, this approach should allowinvestigation of cellular phenotypes for disease states found inconditions that are caused by diverse mutations. At the moment, moststudies utilizing iPSCs for disease modeling have focused on a smallnumber of lines originating from individuals harboring either a singleor small number of highly penetrant mutations. The expanded scale andreduced variation of the automated system should lead to greatlyimproved statistical power in addressing the question of whether amodest effect observed in culture is a direct result of the geneticbackground of the subject in question.

This increased sensitivity should assist in accurately assessing theimpact of common variants that influence human health. Although thesevariants may only contribute modestly to the overall phenotype in agiven individual, their prevalence in populations highlights theirimportance in understanding their role in disease. The ability toaccurately study such variants would represent an important next phasefor in vitro disease models. If there is one common process thateventually leads to cellular dysfunction in each particular disease, itmay be possible to devise a single strategy for inhibiting it, providinga therapeutic for all patients. In contrast, it may be that subsets ofpatients follow distinct paths towards disease. If this is the case, itwill be necessary to identify these distinct groups of patients so thata therapeutic strategy that is specific to the particular path theirdisease follows can be devised and appropriately administered. As thesystem described here is designed to perform standard cell culturemanipulations, adaptation of this robotic technology to directeddifferentiation should further enable the discovery of molecular andgenetic pathways that underlie our traits and disease.

Experimental Procedures Cell Lines

Recruitment of volunteers and biopsy collection and written informedconsent procedures were approved by Western Institutional Review Board.Human embryonic stem cell lines were obtained from the Harvard Stem CellInstitute. Additional reference iPSC lines BC-1, ND1.4 and 2.0 wereobtained from NIH Center for Regenerative Medicine (Chen et al, 2011;Cheng et al., 2012).

Automated Systems Description

To accomplish fibroblast banking, iPSC generation, and iPSCcharacterization and freezing, three integrated robotic platforms wereassembled from a combination of eight automated liquid handlers(Hamilton STAR and STARlet), five incubators (Thermo Cytomat), tworobotic arms (Hamilton RackRunner), four automated Celigo plate imagingsystems (Nexcelom Bioscience), three cryotube decappers (Hamilton),three plate centrifuges (Agilent Vspin), and one plate sealer (AgilentPlateLoc). User initiated software method scripts that communicate withthe relevant instrumentation were written using custom software on theVenus platform (Hamilton) to automate individual process steps describedbelow. Usage of shared automated devices was controlled by a customsoftware reservation system. Disposable conductive pipetting tips(Hamilton) were used throughout processes and tip reuse was minimizedduring all process steps to prevent cross contamination of cultures.Standard plate formats and tubes were used and tracked by barcode.Liquid handling procedures are tracked and logged providing processtraceability.

Automated Biopsy Outgrowth and Fibroblast Cell Culture

Dermal fibroblasts were derived from donor tissue samples collected inbiopsy collection medium (see extended methods below for all mediaformulations). Samples were washed in Biopsy Plating Media, cut into 1-2mm pieces, added to a 6 well plate and dried for 15 minutes after which,biopsy plating medium was added to wells dropwise. Plates were leftundisturbed for 10 days to allow for initial outgrowth before beingtransferred to the robotic system for automated culture and expansion,growth rate analysis, mycoplasma testing and freezing.

Automated Reprogramming

Automated methods were used to thaw and seed fibroblasts onto 12 wellplates (Corning, #3513) then perform medium exchanges every third dayuntil reaching 70-90% confluence. Cells were robotically dissociatedusing TrypLE Select CTS (Life Technologies, #A12859-01), counted byautomated imaging procedure after viability staining using Hoechst(Sigma, #B2261) and Propidium Iodide (Life Technologies, #P3566)followed by cell number calculation for robotic transfer into a Geltrex™(Life Technologies, #A14133) coated 24 well plate (Corning, #3526).Reprogramming of fibroblasts was performed using automated transfectionand feeding methods to deliver modified mRNA (Stemgent, #00-0071) andconditioned Pluriton reprogramming medium supplemented with B18R (200ng/mL) as per manufacturer's instructions. After 15-20 days followingthe initiation of reprogramming, cultures were transitioned to Freedommedia (Life Technologies, #A14577SA) for an additional 5-10 days, afterwhich cells were live stained with TRA-1-60 (Life Technologies, #A13828)to identify iPS colonies by the automated imaging system. Sendai virusinfections were performed using an automated infection protocol withvirus preparations kept cold on a chiller block located within therobotic platform.

Automated iPSC Purification

After reprogramming, iPS cells were enriched by automated depletion ofnon-reprogrammed fibroblasts using a MultiMACS™ system (Miltenyi Biotec,#130-050-601) integrated into a Hamilton STAR liquid handling systemusing anti-human fibroblast microbeads (Miltenyi Biotec, #130-050-601).The iPSCs were collected, seeded into 4 wells of a 96-well imaging plate(BD Biosciences, #353219), and serially diluted 3-fold in adjacentwells. Based upon growth rates calculated from automated imaging, wellconfluency levels and the presence of live TRA-1-60 surface markerexpression, samples were selected and robotically cherry-picked afterbulk dissociation with 0.05 mM EDTA (Life Technologies, #15575-020), andconsolidated into a new Geltrex-coated 96-well plate (Corning, #3599).

Automated iPSC Passaging

Automated methods were used to perform daily feeding and imaging ofconsolidated iPS cell lines using Freedom medium. Upon reaching 70-90%confluence, the cell were passaged using Accutase (Life Technologies,#A11105-01). Growth medium was supplemented with Thiazovivin (1Stemgent, #04-0017) for the first 24 hrs after passaging to promote cellsurvival, after which cells were fed daily with growth medium.

Automated EB Formation

Cells were dissociated and plated for gravity aggregation using theautomated liquid handling systems in 96 well V-bottom plates (Greiner,#651161) in the presence of human ES culture medium without bFGF (seeextended methods for formulation) supplemented with 1 μM Thiazovivin forthe first 24 hrs after passaging. Cell aggregates (EBs) were allowed togrow for a total of 16 days with media refreshed every 48 hrs byautomated methods. EBs were imaged before being harvested and lysedusing automated methods and cell lysate analyzed with custom NanoStringcodesets (Pluri25 and 3GL (Kahler et al., 2013 on the NanostringnCounter system according to manufacturer's instructions.

General Methods

Pluripotency of iPSCs and hESCs was verified by immunofluorescence forthe markers: Nanog (Cell Signalling Technology, #4903), Oct4 (#09-0023),Sox2 (#09-0024), SSEA4 (#09-0006), Tra 1-81 (#09-0011), Tra 1-60(#09-00110; all Stemgent). Images were acquired using a Celigo, NikonEclipse TE 2000-U or Olympus BX41 fluorescent microscopes. Pluripotencywas also analyzed by FACS analysis for CD-13, SSEA4 and Tra 1-60 on anARIA-IIu™ SOU Cell Sorter as previously described (Kahler et al., 2013).DNA and RNA were isolated from cells using High Pure PCR TemplatePreparation kits (Roche, #11796828001), and RNeasy Micro kits (Qiagen,#74004) respectively. DNA/RNA was analyzed using a NanoString nCountersystem following using NanoString's recommended procedures.

Statistical Methods

Statistical analysis was performed using custom R scripts (Team, 2014).Pluripotency/differentiation scores of candidate iPSC lines werequantifued by calculating the median t-score (moderate t-test) ofpluripotent/differentiation markers gene expression in comparison to thedistribution of expression values for a reference set of 15 hESC lines.The previously described Scorecard method (Bock et al., 2011) was usedto measure the differentiation propensity of day 16 EBs formed fromrobotically derived iPSCs and compared against a new reference set of 10established hESC lines in order to maintain consistency of culturingconditions. To quantitate the variance in deriving iPSC lines usingdifferent methods, standard deviation in gene expression for cell lineswas measured and grouped by different derivation methods. The analysisincluded all genes that were designated as markers for pluripotent,endoderm, mesoderm, and ectoderm cell state. To assess the significanceof gene expression variation difference between two cell line groups,the Wilcoxen signed rank test was used.

Extended Experimental Procedures Donor Recruitment and Biopsy Collection

Dermatology patients undergoing a regularly scheduled biopsy andvolunteers from a diverse population were recruited to donate a biopsyfor the generation of induced pluripotent stem cells. Volunteers freefrom bleeding disorders and/or prone to excessive scarring werescheduled to donate a 3 mm punch biopsy at a collaborating dermatologyclinic. Prior to their participation, all participants provided theirwritten informed consent and study approval was obtained from WesternInstitutional Review Board. The samples were taken from an area of thebody to the doctor's discretion, usually the upper arm or leg. Inaddition to the biopsies, health information questionnaires were used tocollect information such as health and medication history, socialhistory and ethnic background. Upon collection, the samples andaccompanying questionnaires were de-identified using a unique ID andreturned to the NYSCF Human Subjects Research (HSR) staff. Theinformation provided within the questionnaires was then transferred bythe HSR staff to Redcap, a password protected database, linking thede-identified data to the anonymous sample ID for the laboratoryresearchers and the samples utilized for the generation of stem celllines.

Automated Systems Description

We designed three integrated robotic platforms that fully automate theiPSC generation and characterization workflow. In brief, cells arehoused in Cytomat incubators and automated method scripts call outplates onto robotic decks for processing. The first platform forfibroblast banking consists of a Hamilton Starlet liquid handler with aplate shuttle directly connecting a Cytomat C24 incubator. Additionaldevices such as a Celigo cell imager, an Agilent Vspin centrifuge, and aHamilton Decapper were integrated to facilitate fibroblast growthtracking, passaging and freezing processes. The second platform for iPSCgeneration is a cluster of three independent liquid handling systemsconnected by a Hamilton Rack Runner robotic arm and rail. This formatallows parallel processing on multiple systems. Each system has beencustomized for its intended purpose with a combination of channelpipettors, plate heaters, shakers, tilters, and cooling modules. Usageof shared automated devices such as the Hamilton Rack Runner, CytomatC48 incubator, Celigo cell imager, Agilent Vspin, and Hamilton Decapperare controlled by a reservation system. The third platform for iPSCcharacterization and banking is a mirror cluster with a slightlydifferent device configuration for optimized 96-well plate handling.

All Hamilton STAR liquid handling systems are contained within NuAireBSL II biosafety cabinets to maintain a sterile operating environmentduring manipulation of cell culture plates. Remaining components areenclosed in a Hepa filtered hood to maintain a sterile operatingenvironment during transportation of cell culture plates between systemsand devices.

Control, scheduling and inventory software integrate with method scriptsfor fully automated operation of the systems. Each method outputsdetailed log and mapping files of processing steps, and Dropcam videomonitoring cameras record system activity. Consumable usage and reagentbarcodes are also automatically tracked on a database.

Automated Biopsy Outgrowth and Fibroblast Cell Culture

Somatic cell lines (dermal fibroblasts) were derived from patient tissuesamples collected at collaborating clinics in Complete M106 media whichcontains Medium 106 (Life Technologies, #M-106-500), 50× Low SerumGrowth Supplement (Life Technologies, #S-003-10) and 100×Antibiotic-Antimycotic (Life Technologies, #154240-062). Samples werede-identified and assigned an internal barcode for tracking identity andpassage number.

Each sample was washed 3 times in Biopsy Plating Media and cleaned witha disposable scalpel and autoclaved forceps to remove blood, fat andepithelial tissue. Biopsy Plating Media contains Knockout™-DMEM (LifeTechnologies #10829-018), 10% FBS (Life Technologies, #100821-147), 2 mMGlutaMAX (Life Technologies, #35050-061), 0.1 mM MEM Non-Essential AminoAcids (Life Technologies, #11140-050), 1× Antibiotic-Antimycotic, 0.1 mM2-Mercaptoethanol (Life Technologies, #21985-023) and 1% Nucleosides(Millipore, #ES-008-D). Depending on initial tissue sample size, 2-3clean 1 mm pieces were transferred to one well of a 6 well tissueculture plate (Corning, #3516) and allowed to dry down for 15 minutes.After drying, 3 mL of biopsy plating media were added dropwise to eachwell containing tissue pieces and placed in a quarantine incubator for10 days to allow for initial outgrowth undisturbed. Plates were thentransferred to an automated incubator (Cytomat, Thermo Fisher) forroutine cell culture on the automated system. All reagents used forautomated methods were assigned internal barcodes encoding mediaaliquots and reagent lot numbers and scanned into individual methods.

Fibroblasts were maintained in Complete M106 media for one week andmonitored by a Celigo automated imager (Nexcelom) for outgrowth beforebeing changed into antibiotic free M106 media for 3 days. A 200 uLaliquot of fibroblast cultured media from each well of a 6 well plate,representing 1 patient sample, was robotically redistributed into a 96well v-Bottom plate (Evergreen, #222-8031) and prepped for mycoplasmatesting on system 2. System 2, a separate liquid handling robot was usedto perform a mycoplasma luminescent assay using the MycoAlert MycoplasmaDetection kit (Lonza, #LT107-318) with the accompanying MycoAlert AssayControl Set (Lonza, #LT07-518) and read on an integrated BioTek SynergyHT imaging system.

Wells that passed mycoplasma detection on system 2 were enzymaticallypassaged using TrypLE CTS (Life Technologies, #A12859-01) into a new 6well daughter plate, keeping source wells separate at a 1:1 ratio onsystem 1. Passaged cells were maintained robotically in Complete M106 onSystem 1 and monitored using the Celigo automated imager for doublingtimes and ideal freezing confluence. Upon reaching confluence, each wellof the daughter plate was enzymatically passaged using TrypLE SelectCTS, pooled and resuspended in 1.5 ml of CTS Syntha-a-Freeze (LifeTechnologies, #A13717-01). Three 500 uL aliquots of the 1.5 mLresuspended cell suspension were transferred robotically into three 2Dbarcoded Matrix tubes (Thermo Scientific, #3741) for cryopreservation.Matrix tubes, within their rack, were placed in a CoolBox™ 96F System(Biocision, #BCS-147). After 24 hours, one of three cryopreserved matrixtubes representing one patient sample, was transferred from the CoolBoxsystem to an automated −80° C. Sample Access Manager (SAM, HamiltonStorage Technologies) where samples are inventoried and selected forreprogramming runs. The sample access manager inventory database allowsfor flexible recall and downstream process batching of tubes forreprogramming based on multiple factors including density, growth ratesand disease group. The remaining two matrix tubes of the same samplewere transferred from the CoolBox system to liquid nitrogen forlong-term storage.

Automated Fibroblast Thawing

Fibroblasts frozen in matrix tubes, stored within the SAM were removedin batches of 20 and manually counted to determine cell number andviability. Cells were manually resuspended into matrix tubes at knowncell numbers, and frozen using the Biocision CoolBox. At the point ofthaw 48 matrix tubes, typically consisting of duplicates of 20 celllines and 8 BJ fibroblast controls, were removed and placed onto System3. Cells were thawed in a 37° C. water bath for 30 seconds, before beingplaced on the robot deck. Upon starting the method tubes were decapped,fibroblast growth medium consisting of DMEM (#11965), 10% FBS, Glutamax,2-Mercaptoethanol (all Life Technologies) was added to each vial,recapped and automatically centrifuged. The supernatant was subsequentlyremoved, and the fibroblasts resuspended in fresh media before beingtransferred to 4, pre-barcoded, 12 well plates (Corning, #3513). Mappingfiles, traced through a centralized monitoring system, wereautomatically generated through the use of user-generated worklists.Cells were automatically transferred, via the RackRunner, to a cytomatwhere cells were housed. Each 12 well plate was fed every three days,with automated imaging occurring at least three times over a 10 daygrowth period.

Automated Cell Seeding (12w to 24w Passaging)

In brief, cells grown in 12 well plates were washed and dissociated withTrypLE Select CTS. Following neutralization with fibroblast growthmedium, 5% (50 μL) of the cell suspension was transferred into a 96 wellBD imaging plate (BD Biosciences, #353219) pre-filled with 50 μL of PBS(Life Technologies, #14190-144) containing 5 μg/mL Hoechst 33342 (Sigma,#B2261) and 1 μg/mL Propidium Iodide (Life Technologies, #P3566). Theimaging plate was centrifuged for 2 minutes, before being subjected to acell count using the Celigo's Dead/Total application. The cell countswere auto-exported with the liquid handling software automaticallycalculating the exact volume of cell suspension required for transferinto daughter wells of a 24 well plate (Corning, #3526). The Dead/Totalcell count and confluence readout were recorded in each method run.Following the passage, cells remaining the in the original 12 well platewere re-fed and allowed to re-expand for downstream DNA isolation.

Automated Geltrex™ Plate Coating

For Geltrex™ plate coating, 1 mL of Geltrex™ was diluted into 99 mL ofpre-chilled DMEM-F12 (Life Technologies, #10565-018) and kept at 4° C.on a module in System 7. Pre-chilled plates in either 96 well or 24 wellplate formats were automatically coated with 100 μL or 500 μL of thepre-chilled Geltrex™ solution respectively. Coated plates were sealedand stored for a maximum for 2 weeks at 4° C. for later use to avoidpremature gelling. Prior to use, plates were incubated at 37° C. for 1hour.

Automated Reprogramming

For initial testing with Sendai virus (Life Technologies, #A1378001), amethod was established to allow automated addition of the Sendai virusto the passaged fibroblasts. Following a medium exchange into freshfibroblast growth medium the virus, kept chilled on a cooling block onsystem 3, was added dropwise into each well of the 24 well plate. Cellswere briefly shaken for 10 seconds, before being returned to the cytomatincubator via the RackRunner. Cells were medium exchanged daily andmonitored for the presence of colonies with automated imaging via theCeligo. For mRNA transfections, an mRNA reprogramming kit (Stemgent,#00-0071) was used. In brief, 4 hours prior to miRNA transfection theday after passaging, cells were equilibrated in PluritonNUFF-conditioned medium (Stemgent) containing 2500× supplement and 200ng/ml B 18R (both supplied with kits). Following equilibration, cellswere transfected with miRNA on days 0 and 4, with mRNA transfectionsoccurring on days 1-10. The miRNA/mRNA mix was robotically added,dropwise, to each well of the 24 well plate on system 3, followed by a10 second shaking to disperse the mRNA mix throughout the well. Eachday, prior to transfection, plates were media exchanged withpre-conditioned Pluriton medium containing supplement and B 18R. After10 transfections, cells were fed for a further 5 days with thepre-conditioned Pluriton media containing the 2500× supplement. Atransition to Freedom media (Life Technologies, #A14577SA) was made with50% medium exchanges over the subsequent 2 days and cells were furthergrown for up to 30 days before being sorted. Since growth of fungi andmycoplasma can be very slow, and the use of antibiotics can mask thepresence of bacteria in cell cultures. The whole process of automatedsomatic cell reprogramming and expansion of the generated iPSC lines hasbeen processed in antibiotic free media. Strict policies wereimplemented on temperature control, and non-reuse of warmed reagents aswell as barcode tracking systems to prevent variations in media andgrowth factor quality to control environmental growth conditions duringculture. Strict guidelines to maintain the sterile conditions of theliquid handling systems and integrated devices were also implemented.

Automated iPS Cell Sorting

The automated iPS cell sorting method was based on a previouslydeveloped FACS method (Kahler et al, 2013, PloS one). In brief, theworklist defined the 24-well source plate to be sorted and the 96-welldestination plates that the sorted iPS cells should be seeded into. The24-well plate was called from the Cytomat, and half of the samples wereprocessed at a time. Cells from 12 wells were dissociated with Accutaseand transferred into half of a 24-deep well harvest plate (E&KScientific, #EK-2053-S). After a 2 minute centrifugation step, thesupernatant was removed, and cell pellets were resuspended with FACSbuffer. 20 μL of human anti-fibroblast magnetic beads (Miltenyi Biotec,#130-050-601) was added to cells, allowed to incubate for 15 minutes,and then washed with FACS buffer to remove the unbound antibody.Following an additional centrifugation, cells were resuspended with 500μL of FACS buffer and applied to a column block on a magnetic separatorsystem (MultiMACS™ Cell24 Separator Plus, Miltenyi Biotec,#130-098-637). 500 μL of FACS buffer was then applied (×3) as washes,resulting in un-reprogrammed fibroblasts staying bound to the column,with reprogrammed cells passing through and collected as a 2 mL volumein a 24-deep well collection plate. The collection plate was centrifugedfor 2 minutes, supernatant was removed, and cell pellets wereresuspended in 400 μL Freedom medium supplemented with 1 uM Thiazovivin(Stemgent, #04-0017). Quadruplicate aliquots of the mixture containing100 ul of cells were seeded into 4 wells of a Geltrex pre-coated,96-well BD black imaging plate and serially diluted over a 3-fold range.The automated method looped through again to process the second half ofthe 24-well source plate.

Automated Cell Consolidation

This Example describes an automated method for consolidating the iPScolonies that passed quality control measures of confluency readout(>=15%), typical human ESC morphology (e.g., cells with large nuclei,small amount of cytoplasm, and form compact monolayer colony) andTRA-1-60 surface marker expression screening (Celigo TumorsphereAnalysis). Wells in 96-well sorted plates were identified, and acherry-picking worklist was created to dictate source and destinationtransfer patterns. Per run, pairs of 96-well source plates were calledfrom the Cytomat and processed together, until the destination plate wasfilled. Selected wells were washed and incubated with 75 μL of 0.05 mMEDTA for 6 minutes. Automated trituration by tips promoted celldissociation, and 100 μL of cell mixtures were transferred into a newGeltrex-coated destination plate. For the first 24 hours, cells werecultured with Freedom medium supplemented with 1 μM Thiazovivin, afterwhich cells were fed with Freedom daily. The task of cherry pickingcells from targeted wells was formulated as a combinatorialoptimization, where the goal was to efficiently select target cloneswith good recovery and without disturbing cell integrity, according toconfluency read and morphological characteristics captured the day afterconsolidation.

Automated iPS Cell Passaging and Expansion

For cell passage of entire 96-well plates, a worklist was created,indicating source and destination plates. All liquid-handling stepsherein occurred in the entire plate at once. The source plate was placedon the deck and cell media was aspirated. Cells were washed once withAccutase before a further addition of 25 μL per well. Accutaseincubation was for 5 minutes at 37° C. on a heated shaker. Cells wereneutralized with 175 μL Freedom media containing 1 μM thiazovivin, andtransferred to an intermediate 96-well V-bottom plate (Evergreen,#222-8031-01V). Cells were centrifuged for 5 minutes at 300 RCF beforesupernatants were aspirated and cells resuspended in Freedom media with1 μM Thiazovivin. Destination plates, previously robotically coated withGeltrex™ and previously robotically pre-processed by removal of Geltrex™suspension and addition of Freedom media with 1 μM thiazovivin, wereretrieved from a Cytomat incubator and placed on the deck. Cellsuspensions were transferred from the intermediate plate to the newdestination plate. Destination plates were returned to the Cytomatincubator.

Automated Cell Freezing (Passage to Cryovials)

A worklist was created, indicating which 96-well plates were to befrozen into 2D barcoded Matrix tubes in Matrix racks. Allliquid-handling steps use a 96-head. Media was aspirated and cells werewashed with 50 μL Accutase before a further addition of 50 μL ofAccutase was added per well and cells were incubated at room temperaturefor 12 minutes. Enzyme neutralization was performed by the addition ofFreedom media containing 1 μM Thiazovivin. Cell suspensions weretransferred to an intermediate 96-well V-bottom plate and centrifugedfor 5 minutes at 300 RCF. The Matrix rack was automatically de-cappedand replaced onto the deck. Supernatants were aspirated and cells wereresuspended in 200 μL Synth-a-Freeze. Cell suspensions were transferredto the Matrix tubes before being re-capped and manually placed into aCoolBox and stored −80° C. before being transferred 24 hours later toliquid nitrogen for long term storage.

Automated Cell Thawing (Thawing of Matrix rack with 96 Matrix Tubes)

A Geltrex coated 96 well plate was retrieved from the Cytomat incubator.Liquid handling steps were performed with a 96-head. Tubes in the Matrixrack were capped and de-capped when necessary. 700 μL of Freedom mediawith 1 μM Thiazovivin was added to each vial. The tubes in the Matrixrack were centrifuged for 5 minutes at 300 RCF. Supernatant was removedand cells were resuspended in 125 μL of Freedom media with thiazovivin;100 μL was transferred to the 96 well plate. The plate was placed in theCytomat incubator. A 10 μL volume of cell suspension remaining in eachtube was used for Dead/Total cell count by the Celigo imager.

Automated EB Formation

Cells were dissociated with Accutase for 5 minutes at 37° C. and platedin suspension into 96 well V-bottom plates (Greiner, #651161) in thepresence of human ES culture media without bFGF and with 1 μMThiazovivin using System 7. Human ES media consists of Knockout™-DMEM(#10829-018), 10% Knockout Serum Replacement (#10828-028), 1% Glutamax(#35050-079), MEM nonessential amino acids (#11140-050), 0.1 mM2-mercaptoethanol (21985-023); All Life Technologies). Cells from oneindividual well were dispensed into 6 daughter wells in a culture volumeof 150 μL/well to create 6 total EBs per starting well. After 24 hours,1004, of media was removed and added fresh media without Thiazovivin.Media exchanges were performed every 48 hours. On day 16, the EBs wereimaged using a Celigo to determine their presence prior to collection bythe liquid handler workstation. EBs were lysed through the addition ofLysis buffer using a Bravo Automated Liquid Handling Platform (AgilentTechnologies). Lysis buffer 2× contained (0.5% N-Lauroylsarcosine Sodiumsalt (Sigma-Aldrich, #61747), 4M Guanidine Thiocyanate (Sigma-Aldrich,#50983), 200 mM 2-mercaptoethanol (Sigma-Aldrich, #63689), 0.02 SodiumCitrate (Sigma-Aldrich, #C8532), 2% DMSO (Sigma-Aldrich, #D2650). Cellextracts were quantified with Quant-iT™ RNA Assay Kit (LifeTechnologies, #Q-33140). Subsequently, 100 ng of cell extract was usedfor gene expression analysis on the NanoString nCounter system followingmanufacturer's protocol. A custom codeset was used which covers 98 genesrepresenting early differentiation markers of the three germ layers(Kahler, D J et al., 2013).

Immunofluorescence Staining

Cell lines, including hES and hiPS were rinsed twice with 1×PBS, fixedwith 4% paraformaldehyde (Santa Cruz, #sc-281692) in PBS for 20 min atroom temperature and permeabilized with PBS containing 0.1% Triton X-100(herein referred to as PBST; Sigma-Aldrich, #T8787) for 30 min.Nonspecific binding sites were blocked by incubation with PBSTcontaining 10% donkey serum (Jackson Labs, #017-000-1210) for 2 hours atroom temperature. Cells were subsequently incubated overnight at 4° C.in PBST containing 10% donkey serum and specific primary antibodies:1:500 anti-Human Oct4 (Stemgent, #09-0023), 1:100 anti-human Nanog (CellSignaling Technologies, #4903), 1:500 anti-Human Sox2 (Stemgent,#09-0024), 1:250 anti-human SSEA4 (Abcam, #ab16287), 1:250 anti-humanTra 1-81 (Stemgent, #09-0010) 1:250 anti-human Tra 1-60 (Stemgent,#09-0010). Following 3 washes in PBS, cells were incubated with one ofthe following secondary antibodies: Alexa Fluor® 488 donkey anti-Mouse(#A-21202; 1:1000 dilution) and Alexa Fluor® 555 donkey anti-rabbit IgG(#A-21428; 1:1000 dilution). After washing twice with 1×PBS, the sampleswere incubated for 10 min with Hoechst (1 μg/ml) in PBS, followed by afinal wash in PBS. Alkaline phosphatase staining was performed accordingto the manufacturer's instructions (SK-5100). Fluorescence images werecaptured with the Celigo, Nikon Eclipse TE 2000-U or Olympus BX41fluorescent microscope.

To determine pluripotency of automated iPSCs, cells were stained forCD-13 (BD Biosciences, #555394; 1:100 dilution), SSEA-4 (BD Bioscience,#560219; 1:100 dilution), Tra 1-60 (BD Bioscience, #560173; 1:100dilution) and DAPI (Life Technologies)(1:15000 dilution). Stained cellswere analyzed on a 5 laser BD Biosciences ARIA-IIu™ SOU Cell Sorter. Theresulting data were analyzed using FlowJo software (Treestar).

DNA Isolation

DNA was isolated from both iPSCs and fibroblasts. Following the passageof cells from a 12 well to a 24 well, the fibroblasts remaining withinthe 12 well plate were robotically cultured for 10-12 days before beingmanually passaged to 6 well plates. Upon reaching ˜90% confluence, asmonitored through the Celigo, each 6-well plate was manually treatedwith TypLE Select CTS and the resulting cell pellet collected in a 96deep well plate (Corning, #3960). The trough was sealed and frozen at−80° C. until DNA extraction. iPSCs were robotically passaged from 96well plates into 24 well plates before being robotically harvested into24 well plates and sealed before being stored at −80° C. DNA isolationfrom the cell pellets was achieved using the High Pure Template PCRTemplate Preparation Kit (Roche, #11796828001) as per the manufacturer'sinstructions with the following modifications: 1) cells were treatedwith 4 μL of RNase (Qiagen, #19101) for 2 minutes whilst resuspended inPBS; 2) DNA was eluted in 30 μL of water.

Cell Line Karyotyping and ID Testing

Cell lines were karyotyped and an identification record of each line wasmade using Nanostring technology. Karyotyping was undertaken using theNanostring nCounter Human Karyotype Panel (Nanostring Technologies, USA)and performed as per the manufacturer's instructions. In brief, theprotocol is as follows: 600 ng of genomic DNA was Alu1 digested at 37°C. for 2 hours, before being denatured at 95° C. for 5 minutes. Toprevent renaturing samples were kept on ice. A total of 300 ng ofAlu1-digested DNA per sample was mixed with hybridization buffer,capture and reporter codes. Following a 16 hour incubation at 65° C.,samples were transferred to a Nanostring Prep station where hybridizedDNA was bound to an imaging cartridge before imaging. Using referencesamples, a copy number was calculated for each chromosome followingnormalization of the data using nSolver (Nanostring Technologies, USA)and Microsoft Excel. The same protocol was used for a proprietarycodeset that allows the identification of genomic repeat elements. Thiscodeset is based upon 28 previously identified Copy Number Polymorphicregions. (Tyson, C. et al. Expansion of a 12-kb VNTR containing theREXO1L1 gene cluster underlies the microscopically visible euchromaticvariant of 8q21.2. Eur J Hum Genet 22, 458-463 (2014)). A dissimilarityscore between a given pair of samples was calculated as the sum ofsquared differences between the samples' normalized, log-transformedprobe values.

Gene expression analysis was performed using either a custom nCountercode set for pluripotency (Pluri25) or a custom nCounter code set forearly differentiation markers into all three germ layers (3GL)previously described in (Kahler. D J et al., 2013). Cell extractcontaining 100 ng of RNA per sample, previously quantified withQuant-iT™ RNA Assay Kit (Life Technologies), was mixed withhybridization buffer, capture and reporter probes. Following a 16 hourincubation at 65° C., samples were transferred to a Nanostring Prepstation, where hybridized fluorescently-labeled RNA was bound to animaging cartridge before imaging. Data was normalized using nSolver(Nanostring Technologies, USA). Clustering was performed using the Rsoftware. R Core Team (2013). R: A language and environment forstatistical computing. R Foundation for Statistical Computing, Vienna,Austria. ISBN 3-900051-07-0.

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While several exemplary embodiments of the invention have beendescribed, the invention is not limited to these embodiments.

1.-20. (canceled)
 21. An induced pluripotent stem cell produced using anautomated system comprising: a cell plating unit for placing cells on aplate; and an induction unit for automated reprogramming of cells bycontacting on the plating unit with reprogramming factors to productiPSCs.
 22. The induced pluripotent stem cell of claim 21, wherein theautomated system further comprises: a sorting unit for selectivelysorting and isolating the iPSCs produced by the induction unit byidentifying iPSC specific markers.
 23. The induced pluripotent stem cellof claim 22, wherein the automated system further comprises: anexpansion unit for expanding the isolation iPSCs, and selecting theexpanded iPSCs.
 24. The induced pluripotent stem cell of claim 21,wherein the automated system further comprises: a freezing unit forfreezing the isolated iPSCs.
 25. The induced pluripotent stem cell ofclaim 21, wherein the automated system further comprises: a confluencycheck unit which checks the confluency of the iPSCs to determine whetherthe iPSCs have a confluency characteristic.
 26. The induced pluripotentstem cell of claim 25, wherein the confluency check unit periodicallychecks the confluency of the iPSCs.
 27. The induced pluripotent stemcell of claim 25, wherein the confluency check unit periodically checksthe confluency of the iPSCs on a more frequent basis over time.
 28. Theinduced pluripotent stem cell of claim 25, wherein the confluency checkunit checks whether the confluency characteristic is within the range of70% to 100%.
 29. The induced pluripotent stem cell of claim 21, whereinthe induction unit uses a viral vector to initiate reprogramming. 30.The induced pluripotent stem cell of claim 29, wherein the inductionunit uses a retrovirus or a Sendai virus to initiate re-programming. 31.The induced pluripotent stem cell of claim 21, wherein the inductionunit uses small molecules, peptides, proteins or nucleic acids toinitiate re-programming.
 32. The induced pluripotent stem cell of claim21, wherein the automated system further comprises: an electroporationunit for electroporating the cells.
 33. The induced pluripotent stemcell of claim 21, wherein the sorting unit comprises a magnetic sortingunit.
 34. The induced pluripotent stem cell of claim 21, wherein theautomated system further comprises: a cell banking unit for obtainingcells used by the plating unit.
 35. The induced pluripotent stem cell ofclaim 34, wherein the banking unit comprises: a biopsy plating unit forplacing biopsies on a plate; an outgrowth and passaging unit for growingcells; and a mycoplasma test unit for testing the presence ofmycoplasma.
 36. The induced pluripotent stem cell of claim 35, whereinthe mycoplasma test unit comprises a glow luminescence testing device.37. The induced pluripotent stem cell of claim 35, wherein the automatedsystem further comprises: a distribution unit for distributing expandedcells; and a storage and retrieval system for storing the cells.
 38. Theinduced pluripotent stem cell of claim 37, wherein the storage andretrieval system freezes the cells.
 39. The induced pluripotent stemcell of claim 21, wherein the cells are somatic cells.
 40. The inducedpluripotent stem cell of claim 39, wherein the somatic cells arefibroblasts.